low cDNA yield from bacterial total RNA. HELP! - (Oct/07/2008 )
Hello,
Okay, here is what I'm trying to accomplish... I want to synthesize cDNA from a total RNA template. I need enough cDNA product for labelling and hybridization on affymetrix GeneChips. My RNA is of very good quality as confirmed with nanodrop and bioanalyzer. Here is my protocol following the guidelines provided by Affymetrix.
MIXA (volume (uL))
RNA (12 ug) --> (19)
Random Primer ([] = 300 ng/µl) (Invitrogen)--> (4.2)
Eukaryotic poly-RNA Control kit (Affymetrix) (second dillution)--> (3)
DEPC H2O--> (23)
TOTAL VOL. (uL)-->(50)
70°C ;10 minutes
25°C ; 10 minutes
Hold at 4°C
Mix-B ( Volume (uL))
MIX-A (RNA/Primer hybridization mix) --> (50.0)
5X 1st Strand Buffer -->(16.6)
100 mM DTT -->(8.3)
10 mM dNTPs -->( 4.2)
SUPERase-In (20 U/µl)-->(2.1)
SuperScript III (200U/µl)-->(2.00)
H2O --> (0)
TOTAL VOL. (uL) --> (83)
25°C; 10 minutes
37°C; 60 minutes
42°C; 60 minutes
Inactivate SuperScript III at 70°C; 15 min
Hold at 4°C
The only thing I changed was the total ammound of SSIII because in the affymetrix manual, it was said to use 25 U/uL of SSIII !!!!!!!!! which seems to be quite too much (or is it???). For instance, Invitrogen says to use 10U/uL of SSIII. Now my problem is that I obtain very low yields of cDNA!!! From 12 ug of total RNA I always get between 0.8 and 1 ug of cDNA which is pretty low. In the affymetrix manual, it said that I should expect about 5 ug of cDNA. After the RT rx I purify my cDNA with QIAGEN kit (minielute). I don't think the qiagen kit is the prob because I tested it with genomic DNA and its ok.
I would greatly appreciate some help!
Okay,
I managed to find a solution. I simply splitted the 10 ug of RNA in 2 x 5 ug and added 3 uL SSIII / tube. After that, I incubated 2h30min at 42degC followed by 10min 70 deg C. With these modifs, I usually obtain about 3 ug of cDNA which is sufficient to proceed with further steps. It seems that the yield of 5-7 ug of cDNA announced by Affymetrix is pretty much optimistic.
Hope this can help someone eventually.