Real Time PCR's linear range depends on a wide variety of factors including the specifications of polmerase used. The product insert for the reagents your using and the user manual for the equipment your using would be the best place to start looking. Real Time PCR can quantify very low quantities of target DNA in a well optimized reaction.
You can take RNA about 1-2 micro gram (if its plant RNA) and convert it into cDNA. Then from the cDNA take 1 micro liter and dilute with 9 micro liter of RNase free water mix it thoroughly and take 5 micro liters from the diluted cDNA for your real time PCR reaction. The dilution of cDNA will remove any impurities remaining while doing reverse transcription reaction of the RNA.
Qiagen usually recommends 100 nano grams of RNA for qPCR. See the website of Qiagen where its explained in detail
I use about 1-10 ng of cDNA (synth. using random primers) in total per well in a 50 uL reaction. At least for my experiments, this results in a good flexibility in target gene quantification to assess both down- and upregulation within the trustworthy range of CT-values. I use this cDNA input for SYBR green as well as for TaqMan assays (4 genes multiplexed). It would be a good idea to run a dilution series of cDNA - as you anyways do for your primer efficiency - e.g. from 100 ng to 100 pg per well to see what concentration gives you to the best outcome in terms of CT values for your specific protocol/machine/etc.
qPCR is a powerful tool but it requires optimized protocols and special attention when it comes to data normalization.
- Badges
- Science method