It is difficult to help without having the exact protocol and experimental details you used. However, try some of my suggestions below to help troubleshoot.

1. Run the reaction (without purification) out on a urea-TBE gel and see what the nucleic acid profile looks like. The A260/A230 is meaningless without seeing what the RNA profile looks like. If you see no nucleic acid corresponding to your RNA, but see a band corresponding to your DNA template, then you probably forgot to add a reagent to your reaction. if you see a smear, then you probably have RNase contamination in one of your reagents. If the latter is true, then I suggest to discard all reagents and use fresh reagents.

2. If you have RNA after the reaction, but after the purification you get no RNA, then make sure you are using the correct reagents such as acid phenol:chloroform. Also, if your A260/A230 ratio is poor, then you probably have a lot of salt contamination in your sample (make sure to wash your RNA pellet 2-3 times with 80% ethanol.

wait, you are using cDNA as the template? Which RNA polymerase are you using? DM me and I can try to help. I personally have never used cDNA as a template for RNA synthesis.

This post is quite confusing (as Kyle Skalenko notes). In vitro transcription uses DNA as a template to make RNA, so lines like "I used the control RNA" or "my RNA does not get amplified" make no sense in this context.

You should start with DNA + free nucleotides, and end up with DNA and your freshly transcribed RNA. Is that what you are expecting/trying to do?

Maybe break down exactly what you're attempting, and what QC steps you've taken, because at the moment it really isn't clear.

Thank you Kyle Skalenko and John Hildyard for your responses.

I did a bad job of explaining the problem within a context. Here is what I am trying to do:

I want to measure the mRNA abundance of a specific gene (GRIN1) in a small population of cells. I am extracting RNA from a small population of cells that have been sorted out of brain tissue, using Qiagen PureLink RNA Mini Kit. Because the RNA yield is low, I am trying to amplify it using Message Amp II kit. According to this kit, I first synthesize cDNA and then use that cDNA for IVT.

I check my samples on Nanodrop after cDNA synthesis and there is a good 260/280 ratio for cDNA (average ~1ug/ul). Then I proceed with IVT using these cDNA samples. After purification, when I check for RNA concentration on NanoDrop, I get a concentration of zero.

At first, I thought the problem might be the RNA that I extracted and initiated the process with because I keep hearing that the quality of the RNA that you start with is very important. Although my RNA has a good 260/280 ratio (~2.2), it has a very low 260/230 (

Well, I wrote a long reply, but researchgate deleted it. Thanks, researchgate!

Basically, do not do what you are doing. You are introducing many, many entirely unpredictable variables which may or may not work (and which in many cases really shouldn't work) in an attempt to answer a fairly simple question.

If your gene is expressed, you should be able to detect it via standard RNA>cDNA>qPCR.

If you cannot detect it that way, you really won't gain anything, especially anything QUANTITATIVE, by in vitro transcription of that cDNA and downstream...whatever with the resultant RNA. It's...frankly it's a slightly insane idea, and really not what IVT is intended to be used for.

Also, do not nanodrop cDNA, it isn't a helpful metric.

How much RNA are you getting, and how clean is it? How much are you using for cDNA synthesis, and how much cDNA are you using in qPCR (if you are using qPCR at all, at this point)?

Thank you for your response John Hildyard

This is a protocol that has been previously used in the lab. Since the RNA extracted from a very small population of cells, it was below detectable levels on NanoDrop. the cDNA synthesized from that amount of RNA was not enough for us to check for the gene of interest plus housekeeping genes. That is why we proceeded with Message Amp.

In the current experiment, I am not bound to a small cell population however, I have to stick to the same protocol for consistency. So, even though I get enough RNA concentration to proceed with cDNA synthesis and directly qPCR, I have to incorporate the IVT here for the sake of consistency.

My RNA concentration on average is 70ng/ul (in total of 40). If by clean you mean the 260/230, it is ~0.7. I am using 10ul of the RNA for cDNA synthesis (so around 700ng). I have not yet proceeded to qPCR but previously, 10ng of cDNA has been used in each qPCR reaction.

I do agree with John Hildyard , but, Noush Ahmadpour , I understand why you need to follow this protocol. I have checked the product manual for the Message Amp II kit and the protocol itself is quite complicated. I personally have not used this protocol, but I have done extensive IVTs with T7 RNAP and purified tons of RNA. That being said, I listed a few suggestions and requests to help troubleshoot this. But I also recommend to contact the company. Make sure none of the reagents went bad (eg. ask the company to check the lot numbers for the kit you are using and have them confirm that nobody else complained about the kit with that particular lot number).

1. Since your lab uses this protocol, is there another gene that your lab has successfully detected? If so, include that as a positive control in your experiment. Personally, I would repeat the experiment with only the successful gene to confirm that the kit works as you would expect.

2. If the kit can detect the control gene, then I would suggest that you run gels on the cDNA (before and after extraction) and RNA (before and after extraction. You need to see what the profile looks like. You have no idea what you are converting and amplifying by only looking at a nanodrop result.

Send me the gel with expected sizes and I can see if the reaction looks ok.

3. Are you use you are extracting, washing, and eluting the cDNA correctly? Maybe excess salts from the wash step could be interfering with the IVT step? I would think this is possible because the control RNA that came with the kit also failed for you.

Summary:

a. I think this is a technical issue. Check kit component to confirm that they did not go bad or contain an RNase.

b. make sure you are preparing the cDNA correctly. Since the control RNA from the kit failed, then either the kit is bad, contaminated, or you are not prepping the material correctly for the next step.

c. Run the control gels. This will be very helpful in troubleshooting this. It can rule out RNase/DNase contamination. Also, if the kit is new and the control RNA fails, then you can complain to the company about a defective kit (send the control gel images to them). The control RNA from the kit should not fail.

Thank you Kyle Skalenko for your detailed response.

By running gels on cDNA before and after extraction, do you mean running the cDNA synthesized with Oligo dT primer before and after purification?

Yes, the lab had previously detected multiple genes using the same protocol.

What I understand from your point about running gels on cDNA is: amplifying a control gene using the cDNA and running the PCR results on the gel and see if they give bands with desired sizes? I am assuming running cDNA on gel would only give a smear.

Also, I am just not sure how running gel on RNA would work?

I will try your tips and get back for some more help!

Thank you again.

Noush Ahmadpour Yes. You are probably right that the cDNA gel would give a smear. I didn't realize it is a polyT oligo. But still worth running the control gel. Likewise, you should run a control gel of the RNA. Sounds good, keep me posted.