Hello everyone, I have been having issues with the cDNA synthesis that I have been doing recently and wanted to check if there is anything that I can do to improve it as my results have not been consistent. I do not follow a specific protocol so I will give mine here: I add 1ug of RNA, 1ul of random 9mers and top of those to 14ul with water incubate at 70oC. Then add 4ul of 5xRT buffer, 1 ul of 10mM dNTPs, and 0.5 ul of RNase Inhibitor and Reverse Transcriptase each (in the case of the control I add water instead of RTase) which amounts to 20 ul in total. After incubating it at 42oC for an hour I incubate it at 95 for 5 mins to inactivate the RTase. I check the quality by doing a PCR using primers for housekeeping genes and also for primers for genes that I am interested in. And these results are not as consistent as I would want them to be. Thank you for your assistance
Regards;
Ege
P.S. I am trying to synthesize 60 ul of cDNA I would also appreciate any tips on this matter as well. Cheers.
you can do cDNA synthesis with this simple protocol - I always use that and I didn't have any problem :
use 1000ng rna and 1ul primer ( oligo dT or random hexamer or etc. ) and add water to 15 ml
set 70 degree for 10 min
then ASAP after end of 10 min move tubes to ice ( this work help primer to connecting stronger with rna )
add 2 ul rt buffer , 1 ul rt , 2ul dNTP and set this times and degrees
25 'c for 10 min
37 'c for 15 min
42 'c for 45 min
75 'c for 10 min
Hi
How do you extract your RNA? Is it from tissues or cells? Have you assessed the quality and quantities of your extracted RNA?
Wishes,
Bassem
Yeah, they are from single Xenopus embryos, so that would be tissues. Yes I have assessed them and only do the cDNA synthesis if they are good (>250 ng/ul in 20ul with A260/280 being over 1.85).
you can do cDNA synthesis with this simple protocol - I always use that and I didn't have any problem :
use 1000ng rna and 1ul primer ( oligo dT or random hexamer or etc. ) and add water to 15 ml
set 70 degree for 10 min
then ASAP after end of 10 min move tubes to ice ( this work help primer to connecting stronger with rna )
add 2 ul rt buffer , 1 ul rt , 2ul dNTP and set this times and degrees
25 'c for 10 min
37 'c for 15 min
42 'c for 45 min
75 'c for 10 min
Dear Ege
1. 70C for 5-10 min is optimised for priming of Poly A tails with Oligo dT
2. If you just prime with random hexamers you will obtain more efficient random priming if you incubate @ 25C for 10 min followed by snap cooling and incubation on ice for 5 min (to stabilise hexamer-RNA binding as mentioned by Reza)
3. Having made your cDNA and heat inactivated take your 20ul reaction and dilute to 100ul before proceeding with PCR: Generally speaking this works at least as well as using undiluted un purified RT product for PCR but can often work better. It is generally assumed that for un purified cDNA this effect is due to dilution of inhibitors in unpurified cDNA that otherwise (on occasions) can undermine PCR
4. Linked to the above and also PCR efficiency, make sure your RNA is of standard and a certain quality, i.e. 260/280nm > 1.8_2.2 and 260/230nM is > 1.0 and ideally > 1.5: Ratio's < 1.0 implies salt contamination and in particular guanidinium salts designed to inhibit RNAses/DNASes that is they bleed through into your resulting RNA (suggested by 260/230 ratio < 1.0) and inhibit RT
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