Hi

What downstream applications do you plan to use your cDNA for, and do you require long cDNAs? Random primers generate shorter cDNA. If it is just for real-time PCR, random primers will be fine and this will not be affected by RNA degradation. Oligo dTs will produce long cDNAs, but RT using Oligo dT will be sensitive to RNA degradation. Whenever I use oligo-dT primers, I first check the integrity of the RNA first using Agilent Bioanalyzer. Hope this helps.

you can use commercial cDNA synthesis kit. first you have to isolate the RNA and then have to follow the manufacturer protocol for cDNA synthesis.

Hello,

As above I would use a kit. oligo dT is better if your gene of interest has a poly A tail, if you pop those search terms in to google scholar I am sure lots of appropriate references will come up. If you are not sure you could do what I do. I use a kit from Promega for reverse transcription and convert 1ug of RNA in to cDNA. I was unsure which to use out of random primers and oligo dts because I was going to look at a selection of genes and some had poly A tails and some not. I used both, a 50-50 mix of the random primer and oligo dT, eg. if 1 reaction requires 1ul of the "primer" use 0.5ul of each and scale up as required. I hope this helps.

use oligo dT (qiagen) - http://www.qiagen.com/products/accessories/turbocapture/oligodtprimers.aspx

and omniscript RT kit (qiagen)- http://www.qiagen.com/Products/Pcr/QiagenReverseTranscriptases/OmniScriptRT.aspx?r=849#Tabs=t0

Hi

What downstream applications do you plan to use your cDNA for, and do you require long cDNAs? Random primers generate shorter cDNA. If it is just for real-time PCR, random primers will be fine and this will not be affected by RNA degradation. Oligo dTs will produce long cDNAs, but RT using Oligo dT will be sensitive to RNA degradation. Whenever I use oligo-dT primers, I first check the integrity of the RNA first using Agilent Bioanalyzer. Hope this helps.

Hi, first you isolate the RNA from your sample, and convert them into cDNA by using quiagen or several commercial available kits are available , bec RNA is highly unstable but it has exon region which codes for original DNA. so from that you can do the up/down regulation whatever you may need.

Why not try both? Usually, oligo-dT yields full-length clones and random primers don't. But if oligo-dT fails, random primers will at least deliver a result... If you have only limited resources, try oligo-dT first.

I recommend you to use super script kit for cDNA synthesis. it works very well. also, you need to think for the RNA isolation. I mean which tissue, time and so on.

we use commercially available applied Biosystems kit. HighCap cDNA RT Kit with RNAse inhib 200Rnx (cod. 4374966). You need 1 ug of RNA to perform the RT reaction. cDNA is of good quality for both semiquantitative and RealTime PCR. Random primers generate shorter cDNA. If it is just for real-time PCR, random primers will be fine and this will not be affected by RNA degradation. Oligo dTs will produce long cDNAs, but RT using Oligo dT will be sensitive to RNA degradation. Whenever I use oligo-dT primers, I first check the integrity of the RNA first using Agilent Bioanalyzer.

As most people said, choice between random primers and oligodT depends on your downstream application. The advice to use mix of both when you are unsure or you want to get both enriched polyadenylated and not adenylated RNA make sense to me. Also if you target specific RNA, you may consider using specific reverse primer or mix it with random primers. If you additionally want to improve yield and length of produced cDNA good idea would be to supplement your reaction with trehalose as described in this paper:

http://www.pnas.org/content/95/2/520.full

and here:

http://www.sciencedirect.com/science/article/pii/S0003269701954740

Of course as many others pointed out the quality of your RNA preparation is the most important factor. In my personal opinion home optimized silica capture method described by Boom at all (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC269651/) is the best ever way to prepare good RNA.

Even when you have good RNA I strongly recommend including RNase inhibitor in your RT mix. Personally I really like Roche Protector RNase Inhibitor (https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10305&catalogId=10304&langId=-1&countryId=pl&forCountryId=pl&productId=3.6.6.1.2.2) and Transcriptor Reverse Transcriptase becouse both are termostable and cDNA can be made at higher temperatures. That really helps with structured RNAs. However these reagents are very expensive. I got also good results with much cheaper kits from Novazym Polska:

http://www.novazym.ehost.pl/esklep4/reverse-transcriptases-in-sets-c-5_26.html?language=en

Again here adding trehalose may help to perform cDNA synthesis at higher temperatres (up to 60C) even with regular MMLV reverse transcriptase. Without trehalose this enzyme is active only up to 42C and optimal is 37C. More details you can find in the PNAS paper- first link I provided in this post.

It depends what you are using the cDNA for. If it is for QPCR to quantify gene expression, oligo dT works well as long as you design your primers in the 3' end of the gene. oligo dT will give you bias at the 3' end, while random hexamers should give you more uniform coverage.

We used SuperScript III one-step RT-PCR syetem (Invitrogen), and TAKARA RT-PCR kit. Both worked well.

I checked the efficiency of using oligodT, randoms primers or oligodTs + random primers using different samples and different transcripts and, al least in my conditions, random primers alone or random primers + oligodTs worked better than oligodTs alone.

Random primers generate shorter cDNA. If it is just for RT-PCR or real-time PCR, random primers will be okay.

Hey what is your downstream application. If you are planing for some transcriptomics analysis it is better to use oligo dT. If you want to characterize or clone some gene in such case it is better to use gene specific primer to synthesis cDNA for the particular region. Ofcourse here the primer should be designed based on gene mRNA/cDNA seqeuence present in NCBI.

If you objective is real time PCR than better you use randome hexamer. Because RT PCR product will be very short so no need to generate long strand of cDNA.

We use Fermentas (Themoscientific) First Strand cDNA Synthesis Kit for cDNA synthesis. Usually, I use oligo dT to analyze mRNA expression by real-time PCR.

I use Fermentas (Themoscientific) First Strand cDNA Synthesis Kit k1622 for cDNA synthesis and use both of them for cDNA synthesis that included: oligo dt primer  1 µl and random hexamer primer 1 µl

Thank you Bahareh, I'm first strand cDNA synthesis kit with random primer and it does give me good results.

I have FFPE samples and I extracted RNA by RNeasy FFPE kit (Qiagen). As I know, using gene specific primers would be a proper way to make cDNA from RNA as extracted RNA is fragmented! 

Do you have any idea in this regard?

Can i used the pcr product from one step cDNA kit as template for qPCR for gene expression study

Hello!

I want to do cDNA synthesis of hepatitis E virus from serum. Would you please suggest me which one will be good, only random primer, or mix of oligo and random primer?

I decided to work with solis biodyne cDNA synthesis mix. But, confusion in primer selection.

Any suggestions regarding this case?

If its for real time-qPCR go for random hexa primers. For cloning, sequencing, expression studies or amplifying longer pcr strands then use oligo dt. Even both at equal proportion can be used for RT-qPCR.

Thank you Bagath Madiajagan

You should use oligo dt for the cDNA synthesis.

Dear Ayodele Jacob Akinyemi

If you use 1ug total RNA, it would be best for result of gene expression than PCR product