Optimisation of total RNA extraction from bovine oocytes and embryos for gene expression studies and effects of cryoprotectants on total RNA extraction

Abstract

Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol® (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n ≈ 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7–2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng μL−1 for MTP, against 46.4 ± 9.5 ng μL−1 from CKP and 47.6 ± 12.9 ng μL−1 for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng μL−1 and 40.0 ± 12.4 ng μL−1, respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to PROH (1.8 vs. 1.2) (p < 0.05). Better results were also observed on the MTP for gene amplification when compared with the other protocols. For gene quantification, the proposed protocol quantified DNMT1 gene with PCR efficiency (0.933) after normalization against GAPDH and SDHA. Amplification and quantification of genes proved specificity and efficiency of the MTP over the other protocols.

Full text

ISSN 00954527, Cytology and Genetics, 2015, Vol. 49, No. 4, pp. 232–239. © Allerton Press, Inc., 2015.
232
1
INTRODUCTION
To study gene expression in samples with small
number of cells and tissues, many different techniques
have been developed. Normally a single mammalian
cell contains 10–30 pg of total RNA out of this around
85% is ribosomal RNA (rRNA), 15–20% is transfer
RNA (tRNA) and 1–5% is messenger RNA (mRNA)
[1]. Noteworthy, 35% of mRNA was found in the
nucleus [2, 3]. In this way, there are several difficulties
in isolation of total RNA from embryos and oocytes
because of limiting quantity of cell and consequently
RNA [4], which in bovine oocytes ranges from 0.7 to
5.3 ng in different stages of oocytes development
[5, 6]. Hence a stabilized protocol is necessary to
1
The article is published in the original.
extract total RNA with standard number of oocytes
instead of using variable amount of bovine oocytes and
embryos and further to analyse the function of
reagents as Guanidinium thiocyanate (GNTC) and
reagents in the commercial kit protocols in total RNA
extraction. A biochemical aspect which plays a major
role in the oocytes growth is the mechanisms of Ca
2+
homoeostasis during growth and maturation [7]. The
increased calcium (Ca
2+
) levels helps to maintain
nucleic acids in aqueous phase instead dissolving in
phenol phase, by coprecipitating with nucleic acids in
precipitation step of isopropanol in RNA extraction
[8]. The quality of oocytes also has to be taken into
consideration as only the good ones have more possi
bility to develop as embryos [9]. As oocytes are nor
mally evaluated by morphological criteria, they are
Optimisation of Total RNA Extraction from Bovine Oocytes
and Embryos for Gene Expression Studies and Effects
of Cryoprotectants on Total RNA Extraction
1
K. C. Pavani
a
, E. E. Baron
a
, M. Faheem
b
, A. Chaveiro
a
, and F. Moreira Da Silva
a
a
University of the Azores, Department of Agrarian Sciences, CITAA, Animal Reproduction, Angra do Heroísmo, Portugal
email: jsilva@uac.pt
b
Animal Production Department, Faculty of Agriculture, Cairo University, Giza, Egypt
Received November 1, 2013
Abstract
—Gene expression is required for understanding bovine oocytes meiotic maturation as well as the
potential of embryonic development. In the present study a standardized reagent protocol for total RNA
extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For
such purpose oocytes (
n
= 795) recovered from about 80 ovaries were divided in three groups: Group 1 mod
ified Trizol
®
(MTP,
n
= 355); Group 2 Guanidinium thiocyanate protocol (GNTC,
n
= 140) and Group 3
Commercial Kit protocol (CKP,
n
= 60). Oocytes belonging to group 1 (
n
= 100) and 3 (
n
= 20) were sub
jected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The
240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization,
and 140 oocytes were vitrified using PROH (
n
= 70) and DMSO (
n
= 70) as cryoprotectants, being then fer
tilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA,
GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced
embryos at the stage of blastocyst (
n
Efficiency of the extraction was further compared. The purity of all sam
ples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and
from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA sam
ples (1.7–2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration
was 127.8 ± 9.3 ng
μ
L
–1
for MTP, against 46.4 ± 9.5 ng
μ
L
–1
from CKP and 47.6 ± 12.9 ng
μ
L
–1
for GNTC
protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concen
tration of 61.3 ± 3.3 ng
μ
L
–1
and 40.0 ± 12.4 ng
μ
L
–1
, respectively for DMSO and PROH. Regarding total
RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to
PROH (1.8 vs. 1.2) (
p
< 0.05). Better results were also observed on the MTP for gene amplification when
compared with the other protocols. For gene quantification, the proposed protocol quantified DNMT1 gene
with PCR efficiency (0.933) after normalization against GAPDH and SDHA. Amplification and quantifica
tion of genes proved specificity and efficiency of the MTP over the other protocols.
Keywords
: total RNA extraction, bovine oocytes and embryos, gene amplification and gene quantification
DOI:
10.3103/S0095452715040076

CYTOLOGY AND GENETICS Vol. 49 No. 4 2015
OPTIMISATION OF TOTAL RNA EXTRACTION 233
considered as good quality if they are compact, with
several layers of cumulus cells and granulosa adhering
to cumulus [10].
There are different manual and kit protocols for the
extraction of total RNA, like GNTC/phenol (Guani
dinium thiocyanate) method, Trizol
®
and different
commercial kit protocols. Trizol
®
reagent is one of
most common chemical solutions used in the extrac
tion of RNA, DNA, and proteins included in many of
the kit products. Guanidinium thiocyanate and chlo
ride are most effective protein denaturants [11].
Guanidinium chloride is a strong inhibitor of ribonu
clease so it is introduced as deproteinization agent for
extraction of RNA by Cox [11]. Later on chloride was
replaced by phenol, to extract ungraded RNA from
ribonuclease rich tissues like pancreas [12], hence
GNTC/Phenol (Guanidinium thiocyanate) method is
specific to large amount of tissues. The guanidinium
method has been used not only in RNA isolation but
also in DNA from eukaryotic cells; however the proto
col to RNA differs from DNA and varies according to
the type of tissues from which nucleic acids are
retrieved [11, 13, 14]. Single step method of RNA
extraction, known as Trizol
®
, was introduced by [15],
which is a chemical combination of guanidinium thio
cyanate, phenol and chloroform, providing high yield
and purity RNA results. Trizol
®
protocol was designed
to eliminate ultracentrifugation step in GNTC/phe
nol and chloride method and it is more specific in
RNA isolation because it maintains the RNA integrity
during tissue homogenization and breaking down cells
components [15, 16]. In all aerobic organisms
SDHA
(Succinate dehydrogenase flavoprotein subunit A)
gene functions as a membrane bound component of
both citric acid cycle and respiratory chain [17]. The
physical and catalytic properties of succinate dehydro
genases are from phylogenetic sources of bovine [17].
GAPDH (glyceraldehyde 3phosphate dehydroge
nase) is also known as G3PDH which catalyses the six
step of glycolysis and helps in breakdown of glucose for
energy and carbon molecules [18]. GAPDH acts as a
link between metabolic state to gene transcription by
moving between cytosol and nucleus [19]. The cellular
location of GAPDH is cytosol since the glycolysis
takes place in cytosol.
GAPDH
gene is highly stable
and constitutive expressed at high levels in most of tis
sues and cells [20]. For this reason
GAPDH
and
SDHA
genes are chosen as reference genes.
DNMT1
gene
(DNA methyltransferase1) was chosen to test the effi
cacy of the protocol, to make the comparison between
the housekeeping genes and normal genes.
In the experiment 1, the aim is to establish a stan
dard protocol for the extraction of total RNA from a
minimum number of fresh and vitrified bovine oocytes
and granulosa cells, enabling the amplification of two
housekeeping genes
SDHA
and
GAPDH
and the
DNMT1
gene. The experiment 2 is performed to test
the efficiency of this protocol over embryos at the blas
tocyst stage through gene quantification of
DNMT1.
MATERIALS AND METHODS
Chemicals
The chemicals and reagents used in the experiment
were obtained from Sigma–Aldrich (USA). All the
RNA extraction chemicals are from Ambion
®
Life
Technologies and all the PCR reaction mixtures are
from Fermentas Company (USA).
Collection of Ovaries
Around 80 to 100 ovaries were obtained from a
local abattoir from adult animals, trimmed of adhering
tissue and transported to the laboratory in Dulbecco’s
phosphate buffered saline (DPBS) at a temperature
ranging from 34 to 37
⎪
C within 2 h post slaughtering.
All ovaries were rinsed once with 70% alcohol and fol
lowed by a wash with fresh DPBS upon arrival at labo
ratory.
Experimental Design
In the present study a renewed protocol was devel
oped aiming to test it aptitude against to CKP and
GNTC protocol in total RNA extraction from fresh
bovine oocytes. Further to evaluate the new protocol,
total RNA extraction was performed in vitrified
oocytes using two cryoprotectants: PROH (1,2 propan
diol) or DMSO (Dimethylsulfoxide) (Experiment 1).
To test the newly developed protocol, total RNA
extraction was also performed in embryos at the blas
tocyst stage followed by gene quantification (Experi
ment 2).
Recovery of Immature Oocytes
Cumulus oocytes complexes (COCs) were col
lected by aspiration from antral follicles (2–8 mm
diameter) with 18 gauge needle. Good quality COCs
(
n
= 795) based on their morphological appearance, in
which covered by at least four layers of compacted
cumulus cells and evenly granulated ooplasm, were
washed twice in TCM199 (Tissue Culture Medium199)
supplemented with 2% FBS (Foetal Bovine Serum),
0.3 mg mL
–1
glutamine and 50
μ
g mL
–1
gentamycin
and randomly assigned for nonvitrified control (fresh
COCs) (
n
= 655) and vitrified (
n
= 140) oocytes
groups.
Immature Oocytes Vitrification
/
Thawing
The vitrification method was based on the protocol
used by [21], with some modification using one of the
two cryoprotectants: PROH or DMSO. Briefly, group
from 20–40 immature oocytes were initially equili
brated for 5 min in holding medium (TCM199
medium with Hepes, supplemented with 1.5 M PROH
or DMSO, 0.1 M sucrose, 20% FBS and 50
μ
g/mL
gentamycin).

234
CYTOLOGY AND GENETICS Vol. 49 No. 4 2015
PAVANI e t al .
After equilibration, oocytes were transferred to vit
rification solution (2 M PROH or DMSO in TCM
199 Hepes medium with 0.1 M sucrose, 20% FBS and
50
μ
g/mL gentamycin) for 30 s at room temperature.
The oocytes were then loaded in a French mini straw
(FMS) and immediately plunged into LN2 for storage.
For thawing, the straws were removed from LN
2
,
held in air for 5 s and transferred quickly to a water
bath at 37
°
C for 30 s. The contents of straws were emp
tied into TCM199 Hepes medium supplemented
with 20% FBS, in a stepwise manner of serial dilution
(0.5, 0.1 and 0 M) of sucrose for 5 min in each step.
After thawing, the oocytes were washed twice in
DEPC (dietilpirocarbonate) water and the granulosa
cells were mechanically separated from the oocytes for
denudation. Then the denuded oocytes and the gran
ulosa cells were stored in RNAse free tubes in –80
°
C.
In Vitro Embryos Development Using Fresh Oocytes
Some of fresh oocytes were subjected to in vitro
maturation, fertilization and embryonic development,
according to [22]. Briefly, washed oocytes were
matured in TCM199 supplemented with 10% FBS,
5
μ
g/mL of FSHLH (“Stimufol”, Belgium), 1
μ
g/mL
estradiol17
β
, 0.15 mg/mL glutamine, 22
μ
g/mL Na
pyruvate and 50
μ
g/mL gentamycin and 20
μ
g/mL of
nystatin. After 24 h of maturation under 5% CO
2
in a
humidified atmosphere at 38.5
°
C, oocytes were placed
for insemination in fertilization TALP medium briefly,
thawed semen were washed three times by centrifuga
tion, twice in spermTALP medium (4 mL for each)
and final washing in IVFTALP medium supple
mented with 10
μ
g/mL heparin, 6 mg/mL BSA
(EFAF), 22
μ
g/mL Napyruvate and 50
μ
g/mL genta
mycin and 20
μ
g/mL of nystatin. After removing the
supernatant, sperm pellet was homogenized with
0.25–0.5 mL of remaining IVFTALP medium for
adjusting the sperm concentration to 1 × 10
6
sperm/mL.
Oocytes and sperm were cultured in 50
μ
L of fertiliza
tion medium (10–15 oocytes/droplet) for 22–24 h at
38.5
°
C in 5% CO
2
in air. Presumptive embryos were
denuded by vortexing, washed and cultured in TCM
199 with Hepes supplemented with 3 mg/ml BSA (Fr.
V), 22
μ
g/mL Napyruvate, 10
μ
g/mL NEAA (MEM,
Nonessential amino acid), 20
μ
L/mL EAA (BME,
Amino acid) and 50
μ
g/mL gentamycin and 20
μ
g/mL
of nystatin in incubator at 38.5
°
C in 5% CO
2
in air.
Cleavage rate was determined after 3 days of fertiliza
tion (day 0) and the embryonic development was eval
uated at day 6 of culture until blastocyst stage. Further
the blastocyst stage samples were washed twice with
DEPC water and stored in RNAse free tubes at –80
°
C.
Total RNA Extraction with Three Different Protocols
The commercial kit protocol (PureLink
®
RNA
Mini Kit) was used according to the fabricant and
samples ranging from 10 to 30 oocytes per tube, gran
ulosa cells and vitrified oocytes had the total RNA
extracted and they were resuspended in 50
μ
L of
DEPCtreated water. The GNTC protocol were pro
cessed with samples ranging from 30 to 60, and total
RNA extraction as follow: (1) 100
μ
L of denaturing
solution (4 M Guanidinium thiocyanate, 0.02 M of
sodium citrate), 0.72
μ
L of 14.4 M betamercaptoeth
anol, 10
μ
L of 2 M sodium acetate (pH 4.0), 100
μ
L of
phenol saturated with water (pH 5.5) and 20
μ
L of
chloroform:isoamyl alcohol are added to the sample
and vortex it for 3 min vigorously; (2) the samples were
centrifuged at 12000 g for 5 min; (3) the upper phase
(aqueous phase) were transferred into the RNAse free
tube; (4) 1
μ
L of 2 mg mL
–1
of glycogen, 100
μ
L of iso
propanol added into each sample and mixed by inver
sion; (5) the samples were centrifuged for 12000 g for
30 min at 4
°
C; (6) the supernatant was removed to a
new tube and the RNA pellet was washed twice with
200
μ
L of 75% ethanol in 0.1% DEPCtreated sterile
water; (7) the pellet was air dried for 15 min and fur
ther dissolved in 20
μ
L of DEPCtreated water.
The MTP developed for total RNA extraction were
pe rfo rmed on sam ple s ra nging f rom 10 t o 70 of o ocytes
per tube, vitrified oocytes and granulosa cells by
(1) adding 100
μ
L of Trizol
®
to the samples, pass it in
the vortex and incubate for 3 min; (2) adding 50
μ
L of
chloroform to the RNAse free tubes and invert them
for 15 s incubating at room temperature for more?
3 min; (3) the samples were centrifuged at 12000 g for
30 min at 4
°
C; (4) the aqueous phase was transferred
in to a new tube; (5) 2.5 volumes of isopropanol added
to the aqueous phase collected; (6) the tubes were cen
trifuged at 12000 g for 30 min at 4
°
C; (7) the superna
tant was discarded and the pellet washed with 150
μ
L
of 70% ethanol and centrifuged at 7,500 g for 5 min;
(8) the pellet was dried in an incubator for 30 min at
37
°
C and further dissolved in 20
μ
L of DEPC water.
All the total RNA samples from the three protocols
were stored at –80
°
C, all these samples were pre
heated at 60
°
C for 5 min and evaluated using a spec
trophotometer (NanoVeu GE Company), based on
the spectrophotometer reading of the samples stated in
Tables 2 and 3 further cDNA synthesis was performed.
cDNA Synthesis
Total RNA samples stored at –80
°
C until were
reverse transcribed into cDNA in a total volume of
20
μ
L, with Revert Aid
TM
H Minus First Strand
cDNA synthesis Kit according to the manufacture’s
protocol [23]. Three micrograms of total RNA were
used for reversed transcribed reaction using an oligo
dT1218 (1.0
μ
L or 500 ng) primer and the same vol
ume was used from the reverse transcription reaction
to perform the qPCR. The first step of the reverse tran
scription was incubation of the RNA samples at 65
°
C
for 5 min with 1.0
μ
L of oligo(dT)
18
Primers and
5.0
μ
L of Nuclease free water. Followed it was added
into each reaction 4.0
μ
L of 5X Reaction Buffer, 1.0
μ
L

CYTOLOGY AND GENETICS Vol. 49 No. 4 2015
OPTIMISATION OF TOTAL RNA EXTRACTION 235
of RiboLock
TM
Inhibitor (20 u/
μ
L), 2.0
μ
l of 10 mM
dNTP mix, and 1.0
μ
L of RevertAid
TM
H Minus
MMuLV Reverse Transcriptase (200 u/
μ
L) to the
incubated samples and all this reaction mixture was
subjected for incubation at 45
°
C for 1 h and 70
°
C for
5 min for reverse transcriptase inactivation. The
cDNA reaction mixture was prepared according to
manufacturer’s protocol [23]. All the reverse tran
scribed (cDNA) samples were treated with RNAse H
(Thermo Scientific) for 30 min at 37
°
C by adding
2.3
μ
L of 10X reaction buffer and 0.7
μ
L of RNAse H,
E. coli
(Fermentas). The cDNA samples were stored in
–20
°
C for further polymerase chain reaction with the
housekeeping and DNMT1 genes.
Gene Amplification
Suitable forward and reverse primers (Table 1) were
designed by using Primer plus 3 software [24] for
GAPDH
(GenBank ref. NM_0010434034.1),
SDHA
(GenBank ref. NM_174178.2) genes and
DNMT1
gene (GenBank ref. NM_182651.2).
The PCR amplification of
GAPDH
,
SDHA
, and
DNMT1
was performed in a total volume of 20
μ
L,
with 1X PCR buffer wi th 3. 0 mM o f MgCl
2
, 250
μ
M of
dNTP mixture (adenine, cytosine, timine and guanine),
10 pmol
μ
L
–1
of each primer (forward and reverse),
1 unit of
Taq
DNA polymerase (Fermentas, Thermo
Scientific), 2.5
μ
L of cDNA and water enough to
complete the total volume. The reaction was done in
35 cycles, with 30 s of denaturation at 94
°
C, 30 s of
annealing at 54
°
C and 45 s of extension at 72
°
C. The
cycles had an initial denaturation at 94
°
C during 3 min,
followed by a final extension at 72
°
C for 10 min and
used for electrophoresis. Briefly, 3% of TAE (Tris ace
tate plus EDTA buffer) agarose gel was prepared with
120
μ
L of 1 × TAE buffer, 3.6 g of agarose, 12
μ
L of
SYBR safe (Invitrogen). The gel loaded with 8
μ
L of
PCR products with 2
μ
L of loading dye solution. As
weight molecular marker, 6
μ
L of 1 Kb DNA ladder
plus (Gene Ruler, Fermentas) was loaded and kept for
gel running for 30 min at 120 volts. Gel photographs
were taken for further analysis with a transiluminator
equipment (UVI tech, UVI Doc) and the intensity of
the bands were measured.
Gene Quantification
Total RNA extraction was conducted in blastocyst
stage by modified Trizol
®
protocol, the spectrophoto
metric reading were taken which was followed by
cDNA synthesis and quantitative realtime PCR
(qRTPCR). The gene quantification protocol was
performed using the ABI Prism 7500 (PE Applied bio
systems) in 96 microwell plates and the Thermo Sci
entific Absolute Blue QPCR SYBR Green Low ROX
Mix (Thermo Scientific ABgene
®
UK). All samples,
including the external standards and nontemplate
control, were run in triplicate. The reaction conditions
had been established through a series of preliminary
optimization experiments including the calibration
curves. Each 25
μ
L final volume reaction contained
1X Blue QPCR SYBR Low ROX (which includes
ThermoStart
TM
DNA polymerase and 3 mM of
MgCl
2
in addition to Blue dye and ROX dye), 1
μ
M of
each forward and reverse primer, water and template
cDNA. Template cDNA corresponds to the stage of
blastocyst samples. Water for a nontemplate control
was included to confirm the absence of contamination
in the reaction mixture. The reaction was initiated by
activation of ThermoStart
TM
DNA polymerase at
Table 1.
Primers used for PCR and realtime PCR in the present study
Gene Sequence (5'–3') Product length, bp
T
m
,
°
C
GAPDH
ForwardGCACAGTCA AGGCAGAGA AC 109 54
ReverseTACTCAGCACCAGCATCACC
SDHA
ForwardCTGCAGAACCTGATGCTTTGTG 188 55
ReverseACGTAGGAGAGCGTGTGCTT
DNMT1
ForwardAGCAATGGGCAGATGTTCCA 268 54
Reverse ATCTCGCGTAGTCTTGGTCG
Table 2.
Spectrophotometric readings of fresh oocytes by
different protocols
Number
of Oocytes
Method
of total RNA
extraction
Purity
260/280 nm
Concentra
tion, ng/
µ
L
30 GNTC 1.175 47
50 GNTC 1.25 104.5
60 GNTC 1.10 59.25
10 MTP 1.50 46.25
20 MTP 1.55 79.2
30 MTP 1.50 152.8
40 MTP 1.66 77.75
70 MTP 1.50 199.2
25 CKP 2.12 26.8
10 CKP 2.63 24.4
25 CKP 2.06 31.2

236
CYTOLOGY AND GENETICS Vol. 49 No. 4 2015
PAVANI e t al .
95
°
C for 15 min, followed by 40 threestep amplifica
tion cycles consisting of 15 s denaturation at 95
°
C, 30 s
at 54
°
C and 30 s at 72
°
C. A final dissociation stage was
run to generate a melting curve for verification of
amplification product specificity. The quantification
was carried out using the comparative cycle threshold
(Ct) method, with the results expressed in relation to
endogenous reference genes and a control group. The
gene quantification was performed on the reference
genes (
GAPDH
and
SDHA
) and
DNMT1
required
gene.
RESULTS
Experiment 1: Efficiency of the MTP
over Kit and GNTC Protocol
All spectrophotometer readings of fresh oocytes
samples by different total RNA extraction protocols
are reported in Table 2 which was subjected to gene
amplification. Basing on the ratio of absorbance at 260
and 280 nm the purity of the samples to different pro
tocols ranged from 1.10 to 1.25 for GNTC protocol;
from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for
the MTP. With the total rna concentration from all
samples (Table 2) for the three protocols an average
was calculated for 30 oocytes, being: 127.8 ± 9.3;
46.4 ± 9.5 and 47.6 ± 12.9 ng
μ
L
–1
respectively for
MTP, CKP and GNTC. Further, to demonstrate
potency of MTP in vitrified oocytes, the total RNA
extraction was performed using this new protocol,
comparing with the CKP only, since the GNTC turn
up the worst results in the first part of the experiment 1.
Spectrophotometer readings notifies that good purity
levels were seen in vitrified oocytes with DMSO (rang
ing from 1.93 to 2.27) when compared with vitrified
PROH oocytes (ranging from 1.47 to 2.08). Samples
after being checked by triplicate spectrophotometric
readings, the total RNA samples were subjected to
CDNA synthesis followed by gene amplification. Typ
ical amplification results of housekeeping genes
(
GAPDH
(109 bp),
SDHA
(188 bp)) and
DNMT1
(268 bp) are shown in Fig. 1 to 4. Figures 1 and 2 rep
resent results from the amplification of fresh oocytes
extracted by GNTC, MTC and CKP. In Fig. 1, sam
ples 1 to 3 of GNTC protocol have a total RNA con
centration of 47.0, 104.5 and 59.25 ng
μ
L
–1
, respectively
for sample 1–3. In samples 4 to 8 of MTP total RNA
concentration was 46.25, 79.2, 199.2, 77.75 ng
μ
L
–1
,
respectively for sample 4–7. In Fig. 2, working with
the CKP, total RNA concentration was 24.4, 31.2,
24.0 and 21.2 ng
μ
L
–1
respectively for samples 1–4.
Further the amplification was performed on
DNMT1
gene (Fig. 4) to test the viability of the MTP
over CKP in general gene instead of housekeeping
gene. The amplification of
DNMT1
gene (268 bp),
with total RNA extracted of fresh oocytes and granu
losa cells using the MTP and CKP are shown in Fig. 3
in which samples 1 to 3 of MTP had total RNA con
centration as follows: sample 1 with granulosa cells
recovered from 10 oocytes having concentration of
104.5 ng
μ
L
–1
, sample 2 of 20 oocytes with concentra
tion of 79.2 ng
μ
L
–1
and sample 3 with 77.75 ng
μ
L
–1
concentration from 40 oocytes. Samples marked as
4 to 7 of CKP had total RNA concentration as follows;
sample 4 of 30 oocytes with concentration of 24.0 ng
μ
L
–1
,
sample 5 with concentration of 21.2 ng
μ
L
–1
from
40 oocytes, sample 6 with granulosa cell collected
from 30 oocytes with concentration of 34.4 ng
μ
L
–1
Table 3.
Total RNA concentration and purity of the blasto
cyst stage samples by using MTP
Embryos
at Blastocysts stage
Purity
260/280 nm
Concentra
tion, g/
µ
L
12 blastocysts fresh 1.81 92
11 blastocysts fresh 1.56 164
11 blastocysts DMSO 1.82 308
12 blastocysts DMSO 1.82 324
9 blastocysts PROH 1.24 288
11 blastocysts PROH 1.26 588
GNTC MTP
200 bp
109 bp
75 bp
M123 4567
Fig. 1.
Amplification of
GAPDH
gene (109 bp) with fresh
oocytes using GNTC and MTP. The numbers 1 to 7 are the
samples: 1 (30 oocytes); 2 (50 oocytes); 3 (60 oocytes);
4 (10 oocytes); 5 (20 oocytes); 6 (70 oocytes); 7 (40 oocytes),
M represent markers.
200 bp
109 bp
75 bp
M
123 4
Fig. 2.
Amplification of
GAPDH
gene (109 bp) with fresh
and vitrified oocytes using CKP. The numbers 1 to 4 are the
samples: 1 (10 oocytes); 2 (25 oocytes); 3 (30 oocytes);
4 (30 oocytes), M represent markers.

CYTOLOGY AND GENETICS Vol. 49 No. 4 2015
OPTIMISATION OF TOTAL RNA EXTRACTION 237
and sample 7 with concentration of 38.0 ng
μ
L
–1
from
granulosa cells recovered from 40 oocytes.
Experiment 2: Productivity of Modified Protocol
in Total RNA Extraction in Embryos
After attaining the results from the experiment 1,
the MTP was evaluated in bovine embryos at the blas
tocyst stage. Table 3, shows that RNA purity ranged
from 1.24 to 1.82 and concentration from 92 to
588 ng/
μ
L. Nonetheless, blastocysts treated with
PROH represented a less purity level of 1.24 and 1.26,
respectively for 9 and 11 blastocysts. Further these
same samples were subjected to cDNA synthesis fol
lowed by real time PCR.
DNMT1
gene PCR efficiency
was 0.93 and after normalization against
GAPDH
and
SDHA
, the relative quantification of this gene was up
regulated in DMSO (1.33) with respect to control and
down regulated in PROH (0.79).
DISCUSSION
After analysing the results of experiment 1 the
developed MTP had proven its efficiency by attaining
better purity and total RNA concentration over other
protocols. From the Table 2 the spectrophotometer
readings of the three protocols, confirm MTP is near
est to the expected purity levels for RNA samples
(1.7–2.0) [25], being CKP’s purity more than 2.0 and
GNTC less than 1.5. As per the total RNA concentra
tion for the average of 30 oocytes representing high
total RNA concentration (127.8 ± 9.3 ng
μ
L
–1
) was
recorded in MTP over the other two protocols. If cal
culations are done for 10 oocytes, it would be possible
to obtain enough total RNA using the MTP and the
CKP (46.25 ng
μ
L
–1
vs. 24.4 ng/
μ
L
–1
) but results
would be null for the GNTC protocol. These results
clearly demonstrate a better performance of MTP and
it would be not possible to work with so few oocytes
using GNTC protocol. Also for the purity levels,
GNTC protocol was the worst and it may be due to
usage of high concentration of different chaotropic
agents (guanidinium thiocyanate, sodium citrate and
phenol) leading to disruption of the three dimensional
structures in macro molecules such as proteins, DNA
and RNA [26]. Research developed by Tsygankova et
al. [27] recommended that after deproteinization pro
cedure of proteins from the “aqueous phase” precipi
tation of nucleic acids can be forced by ethanol and
then to remove impurities, dissolve the resulting pre
cipitate by 2methoxyethanol. Further, the nucleic
acids can be precipitated by cetavlon followed by
washing with 0.01 m sodium acetate by triple redisso
lution of nucleic acids with ethanol. Such procedure
provides absolute purity and nativeness of isolated
preparations of nucleic acids and conservation of their
biological activity from any biological source.
Low total RNA concentration levels were observed
in CKP, possibly of too much washing of materials
with different wash buffer using spin cartridge, where
most of the RNA is clued to their membranes, leading
to the loss of RNA instead of only impurities [28].
Testing the MTP and CKP protocols on vitrified
oocytes had given new result. Usage of DMSO in total
RNA extraction may give better performances when
compared with normal total RNA extraction. Calcu
lation carried out for 30 oocytes, show that, on aver
age, total RNA concentration of DMSO was 61.3 ±
3.3 ng
μ
L
–1
being 40.0 ± 12.4 ng
μ
L
–1
for PROH. This
difference can be due as DMSO accelerates the activ
ity of isopropanol during the precipitation of nucleic
acids extraction procedure [29].
Gene amplification results explains more depth
analysis of the MTP, CKP, and GNTC protocols when
compared with spectrophotometer readings and also
proves how best is the MTP over the other two proto
cols. As it can be observed in Fig. 1 samples 1 to 3 of
GNTC protocol had light bands even though more
number of oocytes (30 to 60) was used during the
extraction. Reason behind for these light bands can be
explained by the poor RNA purity levels of the samples
200 bp
188 bp
109 bp
75 bp
M
1234 1 2 3 4 C(+)C(–)
Fig. 3.
Amplification
SDHA
gene of (188 bp) and
GAPDH
gene with fresh oocytes ranging from 20 to 30 by MTP.
Samples 1 (30 oocytes), 2 (25 oocytes), 3 (20 oocytes),
4 (30 oocytes) with positive (C(+)) and negative control
(C(–)), M represent markers.
200 bp
75 bp
250 bp
300 bp
400 bp
500 bp
MTP CKP
M12 3 4 5
Fig. 4.
Amplification of
DNMT1
gene (268 bp) with fresh
oocytes and granulosa cells by MTP and CKP. The num
bers 1 to 5 are the samples: 1 (granulosa from 10 oocytes);
2(20 oocytes); 3(40 oocytes); 4(30 oocytes); 5(40 oocytes);
M represent markers.

238
CYTOLOGY AND GENETICS Vol. 49 No. 4 2015
PAVANI e t al .
(ratio from 1.10 to 1.25), indicating more DNA and
phenol contamination [30]. The major disadvantage
of GNTC protocol was the usage of larger amounts of
toxic components like phenol, betamercaptoethanol,
and guanidium isothiocyanate reagent, leading to the
contamination of samples affecting thus the RNA
purity levels. Higher amount of phenol leads to less
adsorption of RNA to aqueous phase during the phase
separation process of the RNA extraction. It happens
because less amount of guanidium isothiocyanate
leads to the increase strong repulsion forces between
the negatively charged nucleic acids and the hydroxyl
groups of phenol causing loss of nucleic acids in phe
nol (i.e. more number of nucleic acids resides in phe
nol phase) [31]. The same authors described that
Guanidium isothiocyanate acts as bridge between
phenols, nucleic acids and also decrease the repulsion
forces between them. The other disadvantages of
GNTC protocol was time consuming for preparation as
well as the use of bio hazardous chemicals components.
The amplification results from commercial kit pro
tocol (Fig. 2) had more number of nonspecific bands
for samples (1–4) when compared with PCR amplifi
cation results of other protocols. These nonspecific
bands are maybe due to the required RNA binding to
the walls of spin cartage while washing the samples and
also as the purity levels of some of the samples were
more than 2.0 (i.e. 2.27 and 2.7) showing protein con
tamination. Loss of total RNA material was observed
while transferring the total RNA to new vials and also
some molecules of total RNA can remain in the silica
cartridge with a low volume of washing buffered sup
plied. In the other way if a high amount of washing
buffer is used, it can lead to a high diluted final sample.
Loss of total RNA leads to reduced concentration of
mRNA; hence total RNA concentration levels of CKP
samples were low. Besides being a good protocol for
high amount of tissues, CKP does not work well with
few oocytes being expensive, especially when the total
RNA extraction is not a routine in the laboratory. The
sample 4 (Fig. 1) was amplified with brighter band
even though the oocytes used in total RNA extraction
was very few (10 oocytes), showing the efficiency of
the MTP. Samples 6 (Fig. 1) had shown a nonspecific
band which can be explained by high amount of RNA
concentration 199.2 ng
μ
L
–1
, or by protein contami
1
1
1
nation. As housekeeping genes (
GAPDH
and
SDHA
)
were always being expressed and had indemnity of
amplification, the PCR amplification of these genes,
were evaluated using the total RNA samples isolated
by the MTP. As no nonspecific bands and no negative
control are observed in Fig. 3, it proves that MTP is more
specific to bovine oocytes by obtaining specific bands
with minimum oocyte’s number (i.e. 25–30 oocytes).
After over all analysis of the first part of the experi
ment, GNTC protocol had supposed to be inefficient
towards MTP and CKP, so for the further experimen
tal part only MTP and CKP protocols were studied.
The amplification results of
DNMT1
gene (Fig. 4) had
proven the viability of MTP over CKP. Samples 1 to 3
of MTP had very bright bands when compared with
the samples 4 and 5.
Gene expression of
DNMT1
of blastocysts pro
duced from vitrified oocytes using DMSO as cryopro
tectant shoes up regulation in comparison with control
(C) and down regulated to PROH vitrified ones. After
analysing the results of both experiments (1 and 2) for
the three different protocols, evaluation was done
based on cost, time, and efficiency. Concerning, cost
estimation the purposed protocol revealed to be
cheaper than the other two protocols, being however
very similar to the GNTC protocol. Based the time
consumption, the CKP and MTP was about 3 hours
for each, while for the GNTC protocol, the double
time was necessary. From the efficiency of the three
protocols based on purity, concentration, toxicity of
chemicals and efficiency in gene amplification, the
modified protocol was considered efficient over the
other protocols (Table 4). For bio hazardous chemi
cals, GNTC protocol was much complex when com
pared with MTP and CKP. As far as number of cells is
concerned, the new purposed protocol gives good
results with a minimum of 10 oocytes, while for the
other protocols much more number is required.
In summary proposed protocol here presented can
be considered more effective than GNTC and CKP
after analysing the spectrophotometric, PCR amplifi
cation and gene quantification results. For the total
RNA extraction in bovine oocytes and embryos, the
MTP is more efficient and viable over other protocols.
Table 4.
Efficiency of three protocols in the different conditions
RNA extraction Protocols Modified Trizol protocol GNTC protocol Commercial kit protocol
Safety Less bio hazardous More bio hazardous Less bio hazardous
Efficiency, ng/
µ
L Very good Good Very good
Specificity of bands Bright and specific bands
are observed
Specific band are observed
but light
More nonspecific bandsare
observed
Amount of Tissue
(Fresh oocytes) required 10–30 30–60 25–30

CYTOLOGY AND GENETICS Vol. 49 No. 4 2015
OPTIMISATION OF TOTAL RNA EXTRACTION 239
Project was supported by the Azorean Agency for Sci
ence and Technology, Grant BD M3.1.2/F/044/2011.
CITAA is also fully acknowledged.
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SPELL: 1. guanidium