Hi,

It should be feasible....you can elaborate your query a little bit more..

bye

Hi Ajay,

I have two bacterial totalRNA population that were treated with two different agent and I planing t use TSH (Transcriptome Subtractive Hybridization) method. In this techniques I have to design two different adapters and interestingly one of this adaptor is used also as a cDNA primer. I have attached the paper and If you have time and interested you can check method part of the paper.

Hi Abdugaffar

I have gone through he paper quickly...I feel there are several points in the paper that are either questionable or are not adequately explained...

1) T4-DNA ligase is used for RNA and adapter ligation...In my opinion Ss RNA ligase (eg. T4-RNA ligase should be appropriate here...)

2) adapter and primer sequences are not given...check with the authors or supplementary data

3) RT reaction doenot mention the primer used...the same adapter if ligated to the 3'end of the transcripts ...cannot act as primer...there should be a complementary primer ....

4) authors claim that they have not used endomnuclease...but they uase HaeII in prior to second adapter ligation.....

5) How you'll get rid of 80-90% rRNA in the bacterial RNA preparation.....one can precipiate the rRNA preferentially and then work on the remaining fraction

6) if idea of your experiment is to identify the differentially expressed genes in the two populations...i feel you should for Microarray, NGS based analysis (followed by qRT-PCR validations)....whic have more coverage that SSH....you may loose many genes...in the results....thee approaches are also not so expensive these days

I feel you alo go through some other papers on SSH and modified techniques where adapter sequence are also given ....and then work on the designing the primers and the approach....careful planning will give you good results....do not rush for a quick analysis ..plan a proper analysis

bye