Hi,

We usually use mirneasy from Qiagen to isolate total RNA (with Qiazol, with DNAse treatment). 5 million cells should give a lot of RNA, I can suggest to do QC between steps - clean-up the RNA after isolation, check it on Qubit RNA (do not use NanoDrop if the concentration is low), BioAnalyzer. Check the RIN of your RNA, if it is too degraded, either try the isolation again or use the random priming (instead of poliA) for reverse transcription to do efficient RT on degraded sample. Also check how long is the fragment you amplify on PCR, could be that it is too long.

Good luck,

Michail

Hi there

You can try with Trizol method.

You will get good yield of total RNA.

Good Luck !!

I have two recommendations.

• My go-to: Qiagen RNeasy® Mini (https://www.qiagen.com/us/shop/sample-technologies/rna/total-rna/rneasy-mini-kit/#orderinginformation)
• Machery-Nagel (http://www.mn-net.com/tabid/1311/default.aspx)

You might be able to get a sample from Machery-Nagel by contacting your local area representative.

Hi Gurcharan, I have attached my method for RNA extraction that worked very well for me. It is based on first principles and does not involve a kit. It may be cheeper by using your own reagents and embeds the understanding of the process. Are you going to do reverse transcription for cDNA as I have further methods for this? Hope it helps, good luck!

Thank you all.

Hopefully it works the next time !

I used a Nested PCR system (a modification of PCR) for the cDNA generated from the RNA. This involved designing a second set of primers inside the first fragment used on a second successive PCR run. It's a bit like magic where the first round has no products but they appear after the second PCR with the second primers.

Hi Annwyne Houldsworth,

I have a question for you, "how do you design primers for invisible PCR products to use in second PCR run?" as you suggested for Nested PCR system. thanks.

Good question! You already know the sequence of the fragment for the original PCR amplification and you design a second set of primers. It can be so disheartening to get no products on the first PCR round but you continue as if you have products and miraculously they appear with the second set of primers.

The theory is that, in nested PCR, the second set is intended to amplify a secondary target within the first run product. This allows amplification for a low number of runs in the first round, limiting non-specific products. The more specific second nested set of primers target the fragment and should only amplify the intended product from the first round of amplification and not non-specific product. This allows running more total cycles while minimising non-specific products, useful for very rare templates or PCR with high background. Doing PCR on a sample with apparently no products can seem like a step of faith but is extremely satisfying when it works. Whoever bravely pioneered this is a genius!

I looked at a cell receptor extracted from lymphocytes from patient bloods comparing both genomic DNA and then cDNA, after RNA extraction. The nested PCR produced very acceptable products. I have attached my paper with a table of primer examples.

It is important to understand first principles and know the sequence of the fragment that you are looking for. Hope this helps.

Hi Annwyne

Thank you for your answer, i will apply in my next PCR run.