How much cDNA template (using SuperScriptTM IV CellsDirectTM cDNA Synthesis) is used for a qPCR?

I have a set of experimental RNA samples at low concentrations (55ul each, at 10-25ng/ul concentration range). My end goal is to prepare enough cDNA from these samples to be able to run several Taqman assays to measure expression of a variety of genes. I intend to use Invitrogen SuperScript IV.

Typically, I would use 11ul of RNA at 200ng/ul (i.e. 2.2 ug total RNA) in a 20ul RT reaction; and after the RT add 200ul of water, thus resulting in 220ul of cDNA at 10ng/ul... enough to run over 40 Taqman reactions if 5ul (50ng) is used per reaction.

Given the lower concentration of my current RNA samples, I would like to reverse transcribe all 55ul of my starting RNA, in a larger 5-fold scaled-up RT reaction (100ul total reaction volume). I obviously would have to proportionally scale up my buffer components, but is it necessary to also scale up my Superscript (in order to maintain its final concentration, so that it does not become too dilute in the reaction tube), or is it sufficient to use the same quantity of Superscript, given that the ratio of Superscript:RNA would still be well within the unit-capacity?

In short, which is the more important factor: superscript reaction concentration, or Superscript:RNA ratio? Thanks!

Frank

while using "Nanodrop" for quantification of cDNA, one should quantify it as double-stranded DNA or single-stranded DNA?? I am using Fermentas kit for first standsynthesis of cDNA from total RNA and m not using RNaseH to degrade the RNA

Hello. We have a sample with an unknown concentration of polyphenols. We're diluting the sample in order to perform the Folin Ciocalteau method.

0.2 grams sample / 4 mL 70% methanol and 30% water = initial

200 microliters initial /800 microliters solvent = next dilution

100 microliters initial /900 microliters solvent = final dilution

We performed this several times. However, the final concentration calculated from our standard curve for each dilution was different. Additionally, all the replicates had a general (but not mathematic) trend of increasing concentrations with increasing dilutions. I was wondering where we could be going wrong in our approach and what could be causing this (assuming it's an error in our calculations).

Thanks.

My aim is to create a standard for qPCR for absolute quantification of DNA extracted from soil. The reaction contains PowerUp SYBR Green Master Mix, BSA, primer pair (A189F-MB661R- 0.67 uM), and plasmid DNA.

The qPCR condition is as of from Kolb et al. (2003). The primer A189F is from Holmes et al. (1995), while the mb661r is from Costello & Lidstrom (1999).

I had perform serial dilution up to 7X with triplicates, however, the standard curve result showed a low efficiency of 88.46% and error of 0.244.

I understand that acceptable range for qPCR efficiency is between 90-110%.

How do I improve this to the acceptable range?

can any budy explain how i calculate the DNA concentration for the 50ul reaction?

I have obtained 100 ul of pre-amplified cDNA using the SSIV SINGLE CELL PRE-AMP KIT. Now i want to make a dilution because my samples are limited and i cannot directly use the cDNA. So what dilution should i use and decide on for the cDNA template when using the Applied Biosystems™ TaqMan® Gene Expression Master Mix (Cat. No. 4369016).

I've been using the Qiagen Circulating extraction kit to extract the viral RNA from plasma samples. Further I used an in-house RT step (very similar to SuperScript IV from invitrogen) to get cDNA. Now my problem is that my cDNA is quantitatively as well as qualitatively very poor. Does anyone have suggestion on how to optimize cDNA yields?

I have used a column based kit for isolating the RNA from the insect and plant sample. I got a good quantity of RNA, but in the sample QC for Library preparation, the sample failed due to poor RIN value. The Kit I used is specifically for the isolation of RNA of the highest integrity (RIN > 9) still it got a low RIN value.

Sample QC Details (Tape Station)

Sample - insect Qiaxpert (ng/μl) - 1162.9 A260/280 RATIO - 1.99 QUBIT (ng/μL) - 872 RIN Score - 3.9 COMMENTS (QC PASS/FAIL) - FAIL

Observation - Sample concentration outside the functional range for RINe and the assay; The upper ribosomal fragment has degraded.

We are currently working on a project involving the extraction of DNA and RNA from various types of animal samples, such as whole blood, serum, faeces, etc. The objective is to detect various pathogens through Next-Generation Sequencing (NGS). Our approach for each animal is to combine all the extracted DNA and RNA (converted to cDNA) together (e.g., its DNA from faeces + RNA from faeces + RNA from serum + ...), thus reducing the number of samples to be processed during library preparation.

We have an extraction kit that allows us to either extract DNA and RNA together in a single tube, or extract DNA in one tube and RNA in a different tube. Since our intention is to mix them anyway, we are considering the former option. Nevertheless, we are uncertain whether this will impact the RNA-to-cDNA conversion. Will the presence of DNA affect the conversion process? Additionally, are there any potential effects on the integrity of the DNA? While extracting DNA and RNA together would offer significant benefits in terms of saving time, consumables, and reagents, we will not proceed with this option if it might adversely affect the quality of our extracted DNA or RNA.

Thank you very much for your time and assistance.