Hi!

I suggest you to grow up a large volume of cell culture to obtein more RNA for future more cDNA.

In other hand, I think this paper could help you https://www.researchgate.net/publication/236051504_Transcriptome_analysis_of_the_bloodstream_stage_from_the_parasite_Trypanosoma_vivax?ev=prf_pub

Regards

Article Transcriptome analysis of the bloodstream stage from the par...

Dear Weber,

Yes it is possible to prepare WTA library from double stranded cDNA . But in your case you will get a large no of Ribosomal data after sequencing, as you have prepared cDNA using random hexamer from total RNA.

My advice will be you should scale off your cell and isolate RNA and go for mRNA enrichment followed by cDNA synthesis.

@Lucinda and @Rudra,

thank you for your suggestions.

At the moment I can not repeat all the cell culture experiments. Some of them were very tricky and expensive too.

I wonder, if there is a big problem in amplifying cDNA? Even if there is a lot of translated rRNA it should be possible to amplify the cDNA in a linear pattern to do some qPCRs afterwards. (Imagine how many repetitive elements are amplified in a whole genome amplification setup.)

Maybe somone tried and could share his/her experience.

Hi Alex,

Just my opinion, but this seems like an ill-advised direction. Even if it were technically feasible to amplify 'only small amounts of cDNA left' - which is probably just about doable... why would you want to perform an expensive experiment (I assume you're thinking next-gen sequencing) on such material. If you're deviating substantially from standard practice, and your starting material can't be replicated, how will you trust, interpret, and validate your data?

Hi Nicholas,

thank you for your answer.

No, it is not about NextGen. I only want to do some more qPCRs. Maybe 10 genes, but in triplicate. That would do it for me.

Hi Axel,

Have a look at the SMART technology from Clontech

sorry about the long link.

http://www.clontech.com/GB/Products/cDNA_Synthesis_and_Library_Construction/mRNA_Amplification/SMART_mRNA_Amplification?sitex=10030:22372:US&PEBCL1=Vm9r6LKAlYzK0FveQzQqKC8ydE&PEBCL1_pses=ZG0732492BA37F98DF65E7404F785B43842E2E0523FA8654BE53AE8568D719F765D3F11DA9FE8BAB573582F37430CCDCB1D6CD6CCC4F9110BA

Anna

Hi Anna,

thank you for the link. But this is a kit to amplify mRNA before reverse transcription.

I look for a kit/protocol to amplify already rev.transribed cDNA.

Hi Alex,

Somehow I have ended up following this question unbeknown to me, so I get an email update every time someone tries to answer your question.  I know the answer that you want to hear is that you can do it, and here is the magic tool, however this is not the case unfortunately. If your cDNA is not abundant and doesn't have good genome coverage then you are not going to get good results with WTA or qPCR. Basically both these techniques rely on having good quality starting material in order to get good results. If you try either of these techniques on your material, I have not doubt that you will get some results, but it will be highly unlikely that your results will be accurate, and therefore, what is the point of you spending your hard earned time and money. If you have any more RNA for any of your trials, I would start over with that...If not, save yourself some serious grief, and a bunch of money and cut your loses. I highly recommend not using SSII for your qPCR's, as there are now much better enzymes out there that will give you more even coverage of your genome for qPCR. The SuperScript VILO kit works very well for cDNA synthesis for the purpose of qPCR if you want to stick with Life Tech.

Good Luck

Ron

Hi Danielle,

if I get it right from the manuscript you would suggest to do:

1) second strand cDNA synthesis followed by

2) Nextera library generation.

and than....use a whole genome amplification kit (REPLI-G)?

Hi Axel weber,

Did you found a way? I am also in the same situation, could you let me know.(but I am not planning to perform deep sequencing). For RNA as a starting material I found this 

Look at the Complete SeraMir Exosome RNA Amplification kit (5ml ExoQuick, 20 exoRNA columns, cDNA synthesis and amplification components) at https://www.systembio.com/microrna-research/seramir-exosome-rna-profiling/ordering

/aleem

Dear Weber,

Yes you can go for WTA using your prepared cDNA, but you will get huge reads of rRNA which of no use. Hardly you will get 5% reads of transcripts of your interest. Because mRNA population is only up to 5% in total RNA. rRNA contributes about 85-90% of total population. I am sure you have not depleted the ribosomal RNA before cDNA conversion. Its necessary to enrich the mRNA or do a ribo depletion before preparing cDNA library for WTA.