Hi Supratim,

"If the mRNA has a poly-A 3' tail, then an oligo-dT primer can be used to prime all mRNAs simultaneously.

2) If you only wanted to produce cDNA from a subset of all mRNA, then a sequence-specific primer could be used that wil only bind to one mRNA sequence.

3) If you wanted to produce pieces of cDNA that were scattered all over the mRNA, then you could use a random primer cocktail that would produce cDNA from all mRNAs but the cDNAs would not be full length. The major benefits to random priming are the production of shorter cDNA fragments and increasing the probability that 5' ends of the mRNA would be converted to cDNA. Because reverse transcriptase does not usually reach the 5' end of long mRNAs, random primers can be beneficial"

http://www.bio.davidson.edu/courses/genomics/method/cdnaproduction.html

From Life Technologies, there is more about each type of primers here: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/reverse-transcription/rna-priming-strategies.html

Good luck!

Sequence Specific Primers

SSPs offer the greatest specificity and have been shown to be the most consistent of the primer options for reverse transcription. However, they do not offer the flexibility of oligo(dT) and random primers, meaning that a new cDNA synthesis reaction must be performed for each gene to be studied. This makes sequence-specific primers less than optimal for processing limiting tissue or cell samples.

One-step RT-PCR reactions always employ a gene-specific primer for first-strand cDNA synthesis, while two-step RT-PCR reactions allow for other priming options.

Oligo(dT) Primers

Oligo(dT) primers are a favorite choice for two-step cDNA synthesis reactions because of their specificity for mRNA and because they allow many different targets to be studied from the same cDNA pool. However, because they always initiate reverse transcription at the 3´ end of the transcript, difficult secondary structure may lead to incomplete cDNA synthesis.

Multiple types of oligo(dT) primers are available. Oligo(dT)20 is a homogenous mixture of 20-mer thymidines, while oligo(dT)12–18 is a mixture of 12-mer to 18-mer thymidines. Lastly, anchored oligo(dT) primers are designed to avoid polyA slippage by ensuring that they anneal at the 3´UTR/polyA junction. Choosing the best oligo(dT) primer may depend in part on the temperature of the reverse transcription. More thermostable RTs such as SuperScript® III Reverse Transcriptase may perform better with longer primers, which remain more tightly annealed at elevated temperatures compared to their shorter counterparts.

Oligo(dT) primers are not recommended as the only primer for cDNA synthesis if 18S rRNA is used for normalization in a real-time PCR experiment as the oligo(dt) primer will not anneal.

Random Primers

Random primers are great for synthesizing large pools of cDNA. They are also ideal for non-polyadenylated RNA, such as bacterial RNA, because they anneal throughout the target molecule.

Employing a combination of random and oligo(dT) primers can sometimes increase data quality by combining the benefits of both if used in the same first-strand cDNA synthesis reaction. Random primers are used only in two-step qRT-PCR reactions.

Hi Supratim,

"If the mRNA has a poly-A 3' tail, then an oligo-dT primer can be used to prime all mRNAs simultaneously.

2) If you only wanted to produce cDNA from a subset of all mRNA, then a sequence-specific primer could be used that wil only bind to one mRNA sequence.

3) If you wanted to produce pieces of cDNA that were scattered all over the mRNA, then you could use a random primer cocktail that would produce cDNA from all mRNAs but the cDNAs would not be full length. The major benefits to random priming are the production of shorter cDNA fragments and increasing the probability that 5' ends of the mRNA would be converted to cDNA. Because reverse transcriptase does not usually reach the 5' end of long mRNAs, random primers can be beneficial"

http://www.bio.davidson.edu/courses/genomics/method/cdnaproduction.html

From Life Technologies, there is more about each type of primers here: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/reverse-transcription/rna-priming-strategies.html

Good luck!

Sequence Specific Primers

SSPs offer the greatest specificity and have been shown to be the most consistent of the primer options for reverse transcription. However, they do not offer the flexibility of oligo(dT) and random primers, meaning that a new cDNA synthesis reaction must be performed for each gene to be studied. This makes sequence-specific primers less than optimal for processing limiting tissue or cell samples.

One-step RT-PCR reactions always employ a gene-specific primer for first-strand cDNA synthesis, while two-step RT-PCR reactions allow for other priming options.

Oligo(dT) Primers

Oligo(dT) primers are a favorite choice for two-step cDNA synthesis reactions because of their specificity for mRNA and because they allow many different targets to be studied from the same cDNA pool. However, because they always initiate reverse transcription at the 3´ end of the transcript, difficult secondary structure may lead to incomplete cDNA synthesis.

Multiple types of oligo(dT) primers are available. Oligo(dT)20 is a homogenous mixture of 20-mer thymidines, while oligo(dT)12–18 is a mixture of 12-mer to 18-mer thymidines. Lastly, anchored oligo(dT) primers are designed to avoid polyA slippage by ensuring that they anneal at the 3´UTR/polyA junction. Choosing the best oligo(dT) primer may depend in part on the temperature of the reverse transcription. More thermostable RTs such as SuperScript® III Reverse Transcriptase may perform better with longer primers, which remain more tightly annealed at elevated temperatures compared to their shorter counterparts.

Oligo(dT) primers are not recommended as the only primer for cDNA synthesis if 18S rRNA is used for normalization in a real-time PCR experiment as the oligo(dt) primer will not anneal.

Random Primers

Random primers are great for synthesizing large pools of cDNA. They are also ideal for non-polyadenylated RNA, such as bacterial RNA, because they anneal throughout the target molecule.

Employing a combination of random and oligo(dT) primers can sometimes increase data quality by combining the benefits of both if used in the same first-strand cDNA synthesis reaction. Random primers are used only in two-step qRT-PCR reactions.

Hi,

In addition to the comments above. If u just want to quantify some genes by real time, then random primer is sometimes preferable. Since oligo dT starts at the 3 prime end, if ur Realtime primers are not towards the second half of the mRNA, some long transcripts cannot be quantitatively analyzed (one friend of mine struggled with Realtime for long time due to this unexpected problem) . Obviously if u want to clone something oligo dT should be prefered due to the chance of getting full length transcripts. ANy way in >90% cases both of them should work for you ( I have succesfully cloned genes from random primed cDNAs and quantified genes from oligo-dT cDNAs).

Good luck

Thank you guys for your valued answers. I shall try as per ur experienced guidelines :-)

Amongs all these responses I have a question to make: someone suggested oligo dt someone else random primer. I have a 4000bp cDNA whith wich I am having problems to amplify with specific primers that I designed bringing my restriction site sequence. What have I use to get the amplification product???

Cloning greater than 3000bp cDNA will be a challenge independent of whether u use oligo dT or random primers, especially if the target mRNA is bad expressed. You will have to do many things right to get it to work.  A RNA source with good expression and a better Reverse transcriptase suited for longer transcripts (I know some from Invitrogen) will be most important and once you have it you can try your luck with both oligo dT and random primers.

You can also try amplifying and cloning pieces separately and join them later.

Good Luck

Hi Manoj, amplifying and cloning pieces separately you mean that, starting from the cDNA, I have to amplify piece of 400-500bp with specific primer, until I will cover all the length of my cDNA, and then make a ligation between them???

yes, i mean random primed cdna and trying to clone less than 1500bp fragments. Not ligating fragments, but subcloning after u get them cloned separately. Only If the full length pcr is not working. Good luck.

Manoy sorry but when you say random primers you mean that I have to design specific primers ( two pairs for example) for my cDNA. For example one pair may be that one I am using bringing my sequence for restriction enzyme and another pair can fit in the middle of the cDNA sequence, one 1000 downstream the 5' and the other one 1000bp upstream the 3'. Does i sound good? be more clear as I am not properly an expert. 

I mean random-primer for reverse transcription (to get at least partial cDNA fragments from your large mRNA). Obviously for cloning you will have to do PCR with gene-specific primer pairs (may be 3x separate pairs for amplifying portions of your 4000bp separately). Hope it helps.

Why isn't it possible to do a methylation of the restriction enzymesites in the desired cDNA when you use specific primers for a desired gene in that cDNA? 

I have found the Random Hexamers to produce better yields of cDNA for Real-Time PCR when I compared with GSPs and Oligo(dTs). Oligo(dTs) were quite poor followed by the GSPs.   

I find random hexamers are good for cDNA production and subsequent qPCR performance.

thank you all for sharing your experiences, it is really helpful

I reguarly use Oligo (dT) in combination with random primers. Mark, I use 1/10th of the mentioned concentrations( in protocols) for random primers in that case.

Excellent qPCR data, regardless which gene.