The ratio 260/230 and 260/280 do not give a complete view of the RNA quality. You need to perform a electrophoresis of your RNA to check the presence of 18S and 5S RNA or to check the RIN.

Do you use the "good" time for the RT part of your process. For 12Kb of amplification, you could increase greatly the time of RT. In addition, you could increase the amount of enzyme.

Do you notice that the SSIII works at higher temperature than others RT.

And last, you could try to define a primer, specific of your gene in order to decrease the presence of others cDNA. After, your RT, you could perform a RNase treatment to reduce presence of RNA and increase performance of your long range PCR.

Thanks Stèphane, run the RNA in Bioanalizer is a good suggestion, I will take into account.

I did the RT, increasing the time and the amount of RT-enzyme. I did the RT using three primming trategies Random Hexamers, Ologo(dT) and Gene Specific Primers, I did test by qPCR the positive reverse transcription and I got amplification of a short fragment. The problem is that I am not getting amplification on cDNA while on gDNA I get amplification, do you have any suggestion with working over cDNA? The thermal cycling conditions must be modified because the template is a firts stand? I undesrtand that cDNA is almost the same that gDNA but I am having troubles in this sense.

Thanks...

You could increase the time of RNA denaturation and chilled on ice in order to stabilize this denaturation. In some RT, they specified that the mix before adding RT need to be kept at 0°C. In addition, you could increase the amount of dNTP.

OK, here is the scoop - isolate mRNA from the total RNA. Use RT that can work at elevated temperature - MMLV (SuperScript II) and AMV (if you can find it) and do first strand at 48-50oC. Do not use Rnd6-mers - they will give you very short fragments. I would just use oligo dT, but you could get lucky with specific primers as well. Use one or two sets of nested PCR primers to recover your fragment and don't forget RNAse H treatment before that (or NaOH @ 10 mM). Good luck

I wish I had a good answer for you. That's a large product. I think Stephane's suggestion about denaturation is a good one. A product that big has to have a ton of secondary structure in it. But it always comes down to the primers. Do you need just the ORF, or do you need the whole 3'UTR? You can always play with primers in the UTR until you find one that works. Then get the rest of the UTR using RACE

Hi Stéphane, Petre and Joseph

Thanks a lot for yours suggestions.

I actually am using a denaturation time of 65oC for 15 minutes, and then I chill on ice about 15 minutes more; I could increase to 30 minutes? if necessary…

I am using the Super Script III which work at 55oC (more than the 48-50oC of MMLV or AMV).

I did the RT using three priming strategies: Rnd6-mers, oligo(dT) and a gene specific primer (targeting a segment near to the 3’UTR, but within the coding sequence). When I checked for the positive RT reaction by qPCR, I used primers targeting a segment near to the 5’UTR but within the coding region.

Considering that I got signals of amplification in the qPCR (using the three priming strategies) I assume that Full-Length cDNA were synthesized since the primers used target the beginning portion of the gene. I also include the initial DNase treated RNA and gDNA as positive control, as well as a NTC, to discard gDNA contamination in RNA, to confirm that reaction is working well and to discard reagents contamination.

The point is, cDNA as template is problematic for large amplification? or it is simply a PCR conditions optimization issue?

Other point to take into account is that template at issue is an AT-rich genome. As the cDNA synthesized is a first strand, may be possible that long range PCR is not working in the single strand template and amplification is not successfully. I am really worried and surprised because the long-range PCR is working very very well in gDNA, even in the AT-rich genome but not in the cDNA.

My next step is try adding enhancers additives such as DMSO and Betaine, also to add BSA, since after long range PCR reaction I can visualize like a "condensed gelatinous mass" in the PCR reaction product, when I run in the agarose gel 0.8% it stuck in the well and do not run through the gel.

Good luck! Have you also done gradients on the annealing temperature. I'm amazed sometimes how much a difference only 1 degree can make. Then again, most of what my lab does are much smaller products.

https://www.researchgate.net/post/PCR_product_retains_in_agarose_gel_well_during_electrophoresis-any_thoughts?cp=re153_x_p1&ch=reg&loginT=Zq4ZqL4v7gOpv4XFnImzQFHRnMca4Dzb9A8HTsrvOCQ%2A&pli=1

Hey Ariel, concerning your last remark: I do seem to get PCR products 9kb that stick into the well (see my questionlink above, with gelphoto's).

Could you have a look/answer?!

Regards,

Patrick

I would denature mRNA at 85-90oC for 2-5 min without any buffer (no worries), and I would chill in ice cold water for about 1 min or so before addding other components. You should be able to get product with oligo dT and then PCR with 2-sets of nested primers. If not, then try to split your large gene in two (or three) fragments that you can later join by overlapping PCR or if you have proper unique RE- during the cloning. Good luck.

BTW stick to 48-50oC - that will increase processivity of your SuperScript, and don't forget RNAse H treatment after first strand

Hi Ariel,

Have you got some solutions to your long-range A-T rich amplification? If you have, please share with me coz I'm going through the same procedures and difficulties at the moment. However, with regards to your last proposed way forward, DMSO seems to make things worst in my case. Thanks.

Hi Ongey

Absolutely, as you say DMSO make things worse.

What I did was the following (may be some procedures are basic and you should know them, but I will explain anyway).

Total RNA (Ratio 260/280: 1.9-2.0 and RIN ≥8), was RT using the Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit. I did use this enzyme because my template is 3kb up to 15kb, if you work with shorter templates others HiFi RT enzymes can be used. RNA was prime with Oligo(dT)18. RT-PCR was performed following the RT enzyme manufacturer’s instructions.

Once cDNA was synthesized, the full-length PCR was done using the TaKaRa PrimeSTAR GXL DNA Polymerase, again, this enzyme was used because I was working with long templates. I used as much as I could cDNA, to ensure enough amount in the PCR reaction as only some of the cDNA present in the sample be amplified. The full-length PCR include 5X Buffer (5uL), MG++25uM (1uL), dNTPs 2.5mM (2uL), Fw primer 10uM (2.5uL), Rv primer 10uM (2.5uL), PrimeSTAR GXL Taq (1.25U), BSA 10mg/mL (0.4uL). Thermal cycles was 94ºC-5’, followed by 40 cylces of 98ºC-10’’, 50ºC-3’, 68ºC-15’ and a final hold elongation step of 68ºC-20’.

Depending of the initial amount of your template you will see the bands in the agarose gel. If there is not enough initial amounts of cDNA is possible you cannot obtain visible bands. But is possible the amplified product be present in the final PCR products but in small amounts.

I got some bands moreover I confirmed the presence of the amplified product by a specific qPCR by diluting the amplified product 1/1200.

I hope it work for you... my email [email protected] just in case do you need more details.

 dear petre Ianakiev I need a suggestion as well about my cDNa amplification. I have to amplify a gene of 4000bp with specific primers in order to insert restrction enzyme sequence at the 5' and 3'. I started to RV the RNA and then I tried to amplify the cDNA with the Q5 high fidelity polymarase but I didn't see any band. as above there are larger sequence than this, so I think that is not impossible to amplify a such sequence. Can you or anyone else give me a suggestion? 

Hi Umid,

Unfortunately not. I solved the problem received the gene from another group. However, in a couple of days I will have to do a PCR amplification I think with the same gene and I will let you know.

I was supposing that in my samples the gene of my interest was not present....otherwise I can explain why a stupid PCR didn't work.... You are sure your gene is there.. at a moment what I can suggest is to run first a western to double check the protein so your gene is present.

Hi Ariel and others..

I'm also facing the similar issue. I want to amplify the gene of 1.8kb from plant cDNA. I facing the problem in amplifying the cDNA, where my genomic DNA positive control is working absolutely fine. i'm using the High fidelity Phusion polymerase from Fermentas. i tried using different annealing temperatures, enzyme concentrations, cDNA template concentrations and extension time. If you have any suggestions for me, i will be highly grateful.

Thank you