Hi, there. I need to start experiment from very few amount of total RNA. Is there any way to improve the cDNA yield? Will it work if I extend the incubation time at 48℃ from 30 min to 1 h?
Thanks in advance.
i used 2.5ug of RNa for cDNa synthesis and 42C for 1hr and then 95 fro 5 min. this will give a good yield. even in some kits i checked they are using 42C temperature. So u can have a try. If u have RNA less than 2.5ug U can try with that also. i think i will give a good yield too. just u will have to dilute it less after cDNA synthesis.
i did cDNA synthesis with 150ng RNA also and it gives me good yield. just i dilute it with only 10ul buffer/sterilize water instead of 80ul in above case .
good luck
Pratima
Dear Zheng Wang, I have used several kits for RT-PCR and found that the best (high yield) were INTRON (Korea) and KAPA.
Good luck
Hi, Zheng Wang
Normally all kit have capacity to synthesize cDNA up to 5 microgram mRNA (or total RNA as tamplate).
Reverse transcriptase not going to amplify your cDNA, it will give only same amount of cDNA, how much you will use as template (equivalent or less not more)
As you told you have very less total RNA, so if you want more cDNA so you have to optimize your protocol to isolate more total RNA,
Any way if your aim is to use this cDNA for real time PCR so u will use very less quantity, as we use to make cDNA from 1 to 2 microgram total RNA in 20 micro liter volume , in pcr tube and then we use 30 to 50 time dilution for real time pcr.
I don’t know what is your aim but one thing I can suggest that if u are have very less Total RNA so please use random primer to make cDNA, they will give highest efficient cDNA. good for both real time pcr as well as gene amplification (can utilize total RNA to convert cDNA)
Thank you
all the very best
I used ImProm-IITM Reverse Transcription system,
1. RNA (0.5µg/µl) 3 µl
2. Water treat DEPC 1 µl
3. Oligo(dT)15 1 µl
incubate 70C 5 min
after that mix with master mix of Reverse Transcription
and incubate at
25C 5 min ----->42C 1 h 30min ------>70C 15 min -----.4C
Good luck
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