How can I dilute my RNA sample for cDNA synthesis?

Do you think that 100ng of RNA is sufficient to obtain good Cts in a qPCR?

I have about 100 samples to study the expression of a gene by qPCR. (GADPH is the endogenous control)

I have already extract the RNA from all the samples and i went to nanodrop to quantify them. All of them have different concentrations (ng/uL).

I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul reaction. Thank you!

I have a stock solution of 20 uL which contains 4000 ng of cDNA.

My desired reaction for each PCR is 20 uL reaction and I need a total of 8 uL containing 10 ng of cDNA from my stock solution. What calculations/dilutions would need to be done to obtain that in a 5 uL pipetted from the initial 20 uL stock solution?

Kindly explane importance of cDNA dilution before qPCR and how one can determine best dilution factor for cDNA samples? How it will effect results of qPCR?

In other words, how much quantity of cDNA is recommended in 10ul reaction of qPCR?

I am using the high capacity cdna reverse transcription kit by applied biosystems catalog no 4374967 to make the cDNA from my input RNA of 500ng, my reaction size for RT is 20uL as recommended in the manual. Once I get the cDNA, i do a 20 times dilution of the 20uL to get 400uL and then when i measure this 20 times diluted cDNA i usually get a reading of about 100ng/uL. I then use 1uL of this 20 times diluted cDNA sample (so basially 100ng cDNA) for my qPCR. The problem is the ct value of my housekeeping gene is around 29 unlike around 23 or 24 as stated in the manual. I am wondering therefore, perhaps I might have diluted my cDNA sample too much. So the question is, how many times should I dilute my cDNA after the RT in order to attain a ct value of about 23 or 24 as stated in the manual for 500ng of input RNA (Figure 1. Please refer to link)

I am new to cDNA synthesis and qPCR amplification. I am using taqman miRNA assay to measure Ct value for my target and housekeeping gene. The Ct value for my housekeeping gene in above 33 which is wrong ( normal ct is 18-21). Can someone suggest a way to increase the RNA concentration for cDNA synthesis?

My total volume for the RT reaction is 15 ul; the company recommend 10 ng of total RNA for each 15ul reaction.

Here is what I always do :

1- After the isolation of total RNA, I assess my RNA using nanodrop ( I take 2 ul and put them in the machine). An example for the concentration I got is 116.4 ng/ul.

2- I make a dilution of 2 ng/100 ul ultra pure water (I add 1.7 ul of RNA sample ito 98 ul of water).

3- From the diluted RNA sample I take a 5ul, with 7 ul master mix and 3 ul primer and the I run the RT.

Am I doing this right?

What should be the concentration of RNA used for cDNA synthesis considering the analysis of low expressing genes in the template.

Should the concentration be maximum 1ug or can exceed beyond that?

I have 2 concentrations from different samples, Nanodrop for each are 715.5 ng/ul and 467.5 ng/ul and approximately 2 ul was used for each sample.

I am very confused on how to get and equal concentration/amount from both in order to make cDNA to then use for RT-PCR.

I've looked up the protocol we are using (MMLV RT) but don't see anything on how to actually do the calculations.

I'm really really new and everything I keep reading about converting RNA into equal amounts for cDNA is very confusing and I don't know if there is a general equation available.

Please help. I don't know what to do...