cDNA synthesis

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cDNA synthesis

Post by rose26 » Sat Aug 07, 2010 8:38 am

Hello everybody,

I need your help!
I try to obtain a good yield of cDNA from my total RNA in order to make microarrays.
I work on rice.
I use the double stranded cDNA synthesis kit from Invitrogen (superscript II).
I start with 10µg of total RNA, use oligodT primers and add RNase A to eliminate my rRNA but the yield of cDNA is just 10 ng/µL! :cry:
I need more cDNA for the labelling and hybridation of my microarrays...

Is anyone used this kit?
I have no idea of the yield of cDNA I'm supposed to obtain with 10 µg of starting RNA...

Sorry for my english, I'm French... :wink:

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Post by JackBean » Tue Aug 10, 2010 7:00 am

if you use total RNA, the mRNA forms only small part of it, so your yield can't be much high.

Cis or trans? That's what matters.

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Post by roxanalibe » Thu Aug 19, 2010 9:37 am

recently I used the same protocol (first strand dna synthesis superscript from invitrogen, digest the leftovers of RNA by RNaseH from the kit) and it worked fine for me, because I used 3 times the amounts for the reactions and put 12 ug RNA (i extracted by quiagen kit for total RNA and had a final conc of 3.5 ug/ul in 35 ul; you need to check the integrity of your rna by agilent technology before getting forward).

You can check the cdna by doing a simple actin pcr.

My guess is that you dont use enough material to extract rna. and also you need to work fast and in very strigent conditions (eg, you should not extract rna where you do the maxiprep, the RNAases are everywhere :-))

goood luck

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