The optimal concentration of RNA is always stated by the manufacturer of the reverse transcriptase or cDNA-synthehsis kit.

And yes, the optimal concentration means not the highest you can get, but in balance with the buffer volume, concentration of your RT primer(s) and the capacity of enzyme. Most of the time, the companies invested a lot of money and time in finding out what is best for their client.

My advice: try to standardize the amount of RNA used in reverse trascription and always use the optimal concentration recommened by the manufacturer.

hello Miyu

as talked about in the answer below if you are using a standard RT kit all reverse 

transcriptase cDNA synthesis kits ideally require 0.5ug to 1ug of total RNA for efficient

and reproducible cDNA synthesis

https://www.researchgate.net/post/When_doing_CDNA_conversion_to_RNA_how_much_RNA_is_requiredhel

Hello Miyu

to add to what I said above keep in mind also that the quality of your RNA as well as the quantity of your RNA and also the efficiency of your PCR will also underpin the success of q/RT PCR.  Thus: 

1. Make sure your RNA has a 260/280 ratio of 1.8 to 2.2 and at least if not more important make sure your RNA has a 260/230 ratio of >1.0 and ideally >1.5; this indicates low salt contaminants and in particular low levels of GITC which is present in your lysis buffer or trizol and is designed to denature proteins and kill RNAses but which can also interfere with downstream reactions like PCR

2.  Make sure the 1ug of high quality (260/230 > 1.0) RNA your put into your RT reaction is undegraded:  ideally do this by running 100ng if RNA on an Agilent chip and verifying your RIN number as >8.0.  Alternatively run 100ng of RNA on a formaldehyde agarose gel or at the very least a simple agarose gel but make sure you add 10% formamide to your RNA and heat denature for 3-5 min @ 80-90c to resolve secondary structure and keep your rRNA bands sharp and distinct from degradaded material

3.  Apart from using 1ug of high quality undegraded total RNA I your RT reaction consider using random hexamers plus/ minus Oligo DT and not Oligo dT alone. This will increase the efficiency of your cDNA synthesis and will especially help with amplification of low copy number genes

4.  Apart from efficient RT consider also parameters which keep your PCR reaction efficient; namely 

a.  Keep your amplicons between 100-200bp for qPCR and no more than 500bp for standard PCR.  Very small amplicons in qPCR will keep your priming efficiency above the required 85% especiallyfor low copy number genes

b.  Situate your primers within 1kb of the poly A tail/ Oligo dT priming site especially if performing cDNA synthesis with Oligo dT. alone 

C. Avoid designing primers in a target reg

ion with demonstrable high G/C content and potential secondary structire; do this by screening with a program like m fold :

http://unafold.rna.albany.edu/?q=mfold

In conclusion to perform excellent RT use 1ug of high quality (260/230> 1.0- 2.0) undegraded RNA (RIN >8.0) and perform RT with random hexamers instead of Oligo DT. In addition keep your amplicons short (100-200bp for qPCR and 500bp for standard PCR) conducive to the required high efficiency amplification (>85%) and sit situate your primers in a target region of low secondary structure by screening this region with m fold or an equivalent program tool like IDT

https://www.idtdna.com/pages/scitools

Finallly consider good primer design parameters and in particular minimising primer dimer or self annealing by screening primers with the aforementioned IDT tool. I would also add that screening 2-3 sets of primers by gel based RT PCR; optimising conditions and then taking the best primer pair with those reaction conditions into actual qPCR is the best strategy;  I do this routinely

https://www.researchgate.net/post/Should_I_use_random_primer_or_Oligo_dT_ror_relative_quantification_of_gene_expression_in_Q-PCR

Good luck ! 

In RT q-PCR quality of  RNA of your sample matters a lot  rather its quantity ...If RIN not available to you then OD ratios 260/280 and 260/230 must be good enough for your extracted RNA. RT kit efficiency basically is based on the type of primers used in it like oligo dTs or the random hexamers etc. But still if your RNA quality is bad so even the resultant high cDNA content after the RT step will give you something bizarre during your q-PCR step. So basically the protocol for RNA extraction and your personal technique to perform this counts a lot. 

Thank you all for the helpful information. I understand if we run two step qPCR for RNA samples, it is important to check the RNA concentration before the RT reaction and adjust the concentration into the suitable range where RT reaction occurs. How about when we run RT-qPCR (one step)? If there is not much RNA in a sample which means the RT reaction's efficiency is low, we would get lower Ct value after RT-qPCR than the theoretical value, because of the low efficiency of RT reaction. Is there any way to resolve this problem?

Again, thank you very much for the comments.