no, absolutely.

you have to dilute the high-concentrated RNA samples bringing them to the levels of lower concentrations.

Why would you think of it in the first place. Even if the quantifying machines do give some variation, we always use internal controls to normalize for any differences in the initial amount of the template. So, just start by quantifying your RNA on a trustworthy nanodrop machine, use equal amount of total RNA to synthesize cDNA, and then use it for any purpose with suitable internal controls.

You could use a cDNA synthesis kit designed for very small amounts of RNA. One that comes to mind is the Invitrogen Vilo kit. We routinely isolate total RNA from 100 sorted cells and have good success getting good quality cDNA for qPCR. We don't generally quantify the RNA before cDNA synthesis because we use a dual-color single well qPCR system (the housekeeping gene and target gene are amplified in the same well). But even if you don't do qPCR this way, you could forego the quantification, especially if you are working with tiny amounts of RNA, and use your housekeeping gene in your qPCR to normalize your data.

Just two queries:

1) So these PreAmp Master Mix Kits amplify linearly?

2) QPCR works good only when the template is in its linear range. With such kit how does it works for those already highly abundant (as these are the ones usually adopted as internal controls)?

Hi,

I agree with Cheristopher .you can dilute your samples according to the amount of RNA that you can use. for example if you use 0.5 macrogram of RNA you can calculate whether you are able to use the same amount of all samples(from higher to lower concentration) and after that by using the kit for cDNA synthesise you can prepare cDNA and with the same amount of cDNA, the q-PCR could be done.

good luck

No, when you perform the cDNA synthesis you are suppose to add 1ug of RNA to the reaction, so that all your samples are consistent. In this manner you will be able to amplify out your gene in the downstream PCR.

I found that less input cDNA can actually yield better qPCR cycle results. I compared 20ng vs 200ng (as calculated from the amount of RNA input for the cDNA synthesis) and saw that I got more characteristic good quality curves from using the lower amount, you do not want to overload the qPCR reaction with input as it will suck down your primers early.  If you are getting high cycle detection then look to adjusting the primer concentrations for design better primers.  Additionally, you may want to use a gene specific primer for cDNA synthesis.

It is all said before,

indeed you cannot alter your samples by pre amplifieing some of them and others not.

When you have your RNA, your samples should be brought to the same levels (not your entire sample, but the amount you want to convert in cDNA, keep some RNA for later).

The lowest concentration is determinable for the input of RNA in your cDNA reaction.

Personally I didn't see much difference in adding 1 or 2 µg RNA per CDNA conversion, so I try to standard use  1µg. But if your lowest samples only let you insert 0.5µg, it works as fine. If it is only 1 sample that is really low, discard that one and try to extract the RNA again and see if this time you get a better yield. If more then a couple of samples are that low and you have such a big variation in yield, you have to dilute to the lowest amount.

You can up the amount of RNA that you can insert in your cDNA conversion by changing the concentration of some of your components (in case you use separate components). Make your oligo's dubbel concentrated, so you win maybe 0.5-1µl of extra RNA volume you can insert, use 0.5 x of enzyme instead of 1x, etc. (most enzymes these day's work so good that you can use them at 0.5x instead of 1x without loosing quality. This safes money as well.

Note once you have your cDNA, you have to dilute it at least 2x before you enter it in your qPCR reaction, if you don't the reaction components that are still in the cDNA sample will inhibit your PCR reaction. I use a dilution of around 20x.

So you need a even input of RNA in your cDNA conversion (since you cannot correctly measure DNA levels in cDNA (to contaminated with your reaction components) and then you still need an internal standard to refer for removing the last pipetting errors. Then you can say something about expression level difference.

Indeed all your samples must go through the same handelings, you cannot compare samples that have been treated differently (everything can and will have an effect on your results)

I agree that synthesizing your cDNA from the same amount of RNA is the ideal way. I also had good experience with 150 ng RNA. Maybe you should rather think about why you're getting so different RNA yields from your samples. Are they from different tissues or different amounts of cells? It could be worthwhile to test different RNA isolation kits.

Above suggestion reminds me of one aspect- that , see if it is only one accidental sample with low quantity or is it expected to give low yield. The samples, otherwise yielding more, usually have poor quality as well when accidentally in low quantity. So, although I hope you would have done it, if not, analyze the RNA on gel and then only proceed. 

As far as I can see - yes you can preamplify your cDNA - as long as your not interested in the quantitative results. If you are doing quantification, the MIQE guidelines (Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55:611–622) should be your bible - or other holy book of your choice.

Yes, you could if you use an internal control which is reliable proportionally to your amplification.

Depends on what you are looking at, if it is a viral RNA or another low-copy number target then yes you can, but first, of course you should pre-amplify all samples, not a selection, and secondly, you have to pre-amplify your quantitation standards as well, side by side together with your samples. Do not exceed 15-20 cycles in your 1st PCR, otherwise you will be out of the linear range. Pre-amplification can solve several problems, as it not only boosts sensitivity but also improves the accuracy in the lower range of quantitation - and the results are perfectly quantitative as long as you are in the linear range. And do not forget to include internal control (beta-actin, ribosomal rna, GADPH etc). If you are looking at cellular RNA, I would indeed better increase the cDNA yield or RNA isolation efficiency.

Ideally the amplification before qPCR is not recommended because it will alter the the number of templates.

rather you can equalize the sample concentration by diluting the high concentration samples and then take equal amount of templates for qPCR. lower concentration of templates give better results and amplifications in qPCR.

Something not mentioned in these responses is the efficiency of the reverse transcription reaction, which can vary dramatically as a function of total RNA in the reaction. Others have shown--and we have recently confirmed with our own experiments-- that addition of background RNA up to at least 500 ng per reaction (we used 1 µg) improves the yield of target cDNA. We also found that adding much higher primer concentrations above the typical recommendation in the reverse transcription kits (whether gene-specific or N6), also increases the efficiency of the reverse transcription. So, even if uniform pre-amplification could be accomplished (possible, but uncertain, you would need an internal control, but even that is no guarantee), your results could still be wrong if you had varying RNA concentrations in the cDNA synthesis step. Your safest course would be (as mentioned above) to redo the cDNA synthesis step with equal amounts of sample RNA plus background RNA (some RNA source you know will not have your target in it) added to bring all of them up to 1 µg of total RNA. Ideally, you would also first run some titration tests with your primers to find out what the best concentration is for your target (we had to go up to >100 µM with N6 primers or 50 µM with gene-specific to get the most efficient yields from the reverse transcription). When you transfer the cDNA into the qPCR reaction, you may or may not need to an extra dilution ...some kits tolerate the carryover from the cDNA synthesis reaction better than others.

I totally agree to Greg's response. First, pre-amplification kits (whether for DNA or RNA) amplify non-uniformly. And second, There lies a practical reason in using sufficient amount of RNA to start with, say your target RNA is in 1/100000 frequency and only 75000 (presume) molecules made up for 10ng input RNA for cDNA amplification or even pre-amplification kit, do you expect your target RNA will enrich? You still are left on ground Zero. So the ultimate key is to start with sufficient RNA, may be you will have to start from scratch but you will start afresh and start good.