Hello
I am working on the real time PCR for quantifying gene expression levels. Since the conversion rate of RNA to cDNA depends on reverse transcriptase efficiency which things to be consider for the starting material whether RNA or cdna concentration?
In addition to Ali's comments above, ultimately, you will be generating relative numbers by normalizing the signal using an invariable (relatively speaking) house-keeping protein/RNA. This will take care of much of the sample-to-sample starting amount variabilities as long as all else remains the same and you are well-within the reliable portion of quantitation.
An alternative is to perform an absolute quantitation but if you do not already have access to the newer equipment with this capability, it will entails purchasing a newer and not-so-inexpensive machine.
I agree with Ali and Tausif. Should have atleast three replicates to reduce error rate. Make sure you are selecting a suitable reference gene/ may be specific sometimes i dont usually prefer universal reference genes. If you are new to this field you can follow the universal guidelines available for quantitative PCR. If you go for publication your real time PCR should meet this standards. you can just google it
Measuring the cDNA concentration is not plausible. Usually one relies on the RNA concentration, assuming the RT efficiency is the same for all samples. Having 3-4 replicates will help you determine the error rate and the having atleast 2 housekeeping genes should help you normalize your qPCR results
you have to measure the concentration of the RNA before of RT-PCR and the cDNA concentration after of the reaction before of qPCR. There are a lot of variants that can change the final concentration
I agree with everything already mentioned. Have three bio replicates and three technical reps. I also tend to use the same mass of starting material and put the same concentration of total RNA into the cDNA synthesis reaction. Also, find a good reference gene to use as a control. This paper (Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R (2005) Genome-wide identification and testing of superior
reference genes for transcript normalization in Arabidopsis.
Plant Physiol 139:5–17) has good examples for reference genes.
Measuring RNA concentration and quality (260/280, 230/260 ratios) is a good starting point. cDNA concentration is not meaningful. For your gene of interest, relative expression levels will depend on several factors i.e. if you use random hexamers, oligodT primers, your amplicon is 3' or 5' etc. Thus, we use the same amount of starting RNA for all samples, same RT kit, same dilution. Minimum 3 biological replicates, better 6 (higher number increases the reliability for statistic testing) and normalisation to at least three housekeeping genes.
"For your gene of interest, relative expression levels will depend on several factors i.e. if you use random hexamers, oligodT primers, your amplicon is 3' or 5' etc."
Not really, the relative expression levels of any gene will entirely be a function of cellular events and conditions. Accurate determinations of these levels will be effected by inherent limitations of methods employed.
Literature seems to be full of usage of a variety of "house-keeping" mRNA and rather than using 3 or even a larger number of these transcripts and going with average, it is more important to understand which transcripts are invariable under the set of test and control conditions in a given system. Picking house-keeping genes simply because others have used them in their system is not the best strategy for accuracy in measurement of relative expression of any gene of one's interest.
Addendum: I see that I have a very shy admirer! ;)
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