Hi Virginie

In my opinion both the procedures will offer similar quality templates for use in qRT-PCR.......Old fashion protocol are still useful if you want some 'FLEXIBILITY' in execution of experiments and if you can spare time since they take more time and they save 'MONEY'.....since all the steps and components are known and their functions in the protocol are well established..old fashioned protocols can be modified by replacing similar components..

On the contrary kit based protocols can save considerable time but on the expense of substantial amount of 'MONEY' ........ but many components are not disclosed so 'CUSTOMIZATION' if needed is not possible

Hi Virginie

In my opinion both the procedures will offer similar quality templates for use in qRT-PCR.......Old fashion protocol are still useful if you want some 'FLEXIBILITY' in execution of experiments and if you can spare time since they take more time and they save 'MONEY'.....since all the steps and components are known and their functions in the protocol are well established..old fashioned protocols can be modified by replacing similar components..

On the contrary kit based protocols can save considerable time but on the expense of substantial amount of 'MONEY' ........ but many components are not disclosed so 'CUSTOMIZATION' if needed is not possible

Hi Virginie,

I agree with Ajay, Old fashioned kits are better and have more flexibility and control over the reaction as compared to the kits. I personally prefer to do a Trizol extraction followed by DNA digestion step for RNA. It is working like a charm for me till now. It is fast and clean too.

Good luck

Hello Virginie,

I have used both the method and I ll recommend to use kit. Since, it is costlier than traditional method but it is time saving and giving comparatively good results.

Good luck

It also depends on the source tissue. Bone, tendon and muscle tissue provide a lot less mRNA from kits. Therefore, an internally optimized mRNA collection is better. From leukocytes or liver (and the like), the mRNA kits work well and save time. They are consistent and yield high quality results most often. We use cDNA kits once we have good quality mRNA and from there get consistent qRT-PCR using SybrGreen with validated primers .

heyy

I use TaqMan probes and Qiagen kit. are u using it for mirna or RNA? i have altered protocol to isolate differently from protocol.

I agree with the comments above. The quality of RNA ( DNA -free as assessed by O.D., Gel electrophoresis,chip etc) is very important.

Why old fashion protocol???????? All Kits are based on traditional methods. There aren't substantial changes in the protocols from 1980. Using traditional methods you get much more DNA/RNA than Kit.

I agree with David about the type of the sample used. In my lab we use old protocols when working with tissues such as liver and colon. However, when using cells differentiated from stem cells we use commercial kits for extraction of mRNA and cDNA preparation.

Guess it's all said. In case you got difficult tissues to extract from, old school offers more options to adapt. However this might cause concerns in respect of reproducibility, which is allegedly better when using a standardized kit.

if you do cell culture and have many samples kit would save a lot of time. Otherwise this is the matter of what you can afford.

Now a days researchers are using one step RT-PCR kits which allows direct reactions from tissues. This is better if you are planning to go for single reaction. But if you need subsequent reactions from the same template, then you have to go with tarditional method and have to make cDNA, which will be used as template for further reactions. These methods are depend on your experiments, so decide accordingly.

Goodluck.

If something is good for everything, it is not good for anything. Well, if it works, great, but if it doesn't work, not too much room for modifications. It all depends what kind of cells you are working with.

It all depends on what type of RNA you're trying to isolate, i.e., more fresh or flash-frozen vs. archival tissue w/ a lot of protein stuck to it, as well as the region(s) you're trying to amplify, and how big your amplified product is. Therefore, for some samples, good old classical isolations, cDNA transcr. work better. For high-throughput, fresher samples like cell preps, kits (Qiagen, Promega) might be perfect.

Old fascino is cheaper and has higher yields, although you may extract with a tad more of impurities and it takes longer to prepare the reagents. Kits are quicker, expensive, less yield but higher quality of the extract.

Regards.

For qRT-PCR I use RNA extracted with Trizol and DNAseI-treated with the Ambion kit. Use equal amounts for RT with Superscript II (Invitrogen). The transcripts are again evaluated by NanoDrop and diluted to working solutions as templates for the qRT-PCR. Works just fine.

The experience in our lab has shown that the utilization of commercial kit for this kind of protocol is accessible, comfortable and accurate.

The old protocol could be possibly useful to standardize the conditions. But kit provides you accuracy and easy way to execute. Old one is time consuming and kit method is rapid way..simple..

Working with RNA especially mRNA I would rather go for a kit altough phenol containing extraction reagents (e.g.Trizol) is still the best for double stranded RNA.

I recommend the old fashioned method for RT PCR

For qRT-PCR on bacteria I recommend QIAGEN extraction, quick and clean.

I recomend working with kit.

With a kit

You can also try doing 2nd PCR with same primers using first PCR product as template.

I have compared Cells to Ct kit (Applied Biosystems) with the clasical RNA purification, RT, qPCR.....and the results were comparable. Primers for qPCR were from Applied Biosystems (Assay on demand), too. I recommend this kit.

I agree with all above comments. In my experience (working with CNS tissues), the kit has worked fine in terms of mRNA quality and quantity, simplicity and time. However, I acknowledge that kits are expensive so it depends on your budget.

I agree with the comments from Ajay and David and it really depends on your requirements. The original methods i.e. Trizol have much more flexibility so can be tailored more easily to your specific needs both in the extraction method as well as for downstream applications. They also work out cheaper but on the downside they can take longer and are more prone to human error, particularly if you are inexperienced and have a large number of samples to go through. Where the original methods really come into their own is when you are extracting RNA from sources with lots of matrix, polysaccharides and proteoglycans etc. The kits often struggle with such samples whereas additional steps can be used to improve the RNA quality and yield with the original methods. So if you are inexperienced, have lots of samples to go through and are using say cells grown in culture then it is definitely worth spending the extra on the kits. However, if you are more confident and/or have to extract from more difficult tissues/sources then the original method will pay dividends.

I would prefer isolation RNA first instead of using cell lysates. The duplex qPCR with target and endogenous control in the same well gives more accurate results using taqman assay.

I agree with comments from Ajay, David and Ben. Also in my opinion using original methods is difficult and more prone to human error, but it helps U to be expert and Know different aspect of the process and probable mistakes, But if U have many samples and U need an early result or U don't have enough time to spend in learning the concepts of your experiment, kit might be more helpful.

Good luck!

old fashion procedures are still of value in certain part of the world. what ever procedure you are using will depend on your experience and intellegent thinking. Cost is always an issue in developing counteries unless you have a grant.

what ever your situation you will find the proper way to handle your problem

I agree with comments above. The Kit is very useful if you have limited amount of sample. The kit works down to100 cells in our hand. And you can easily handle 96 samples parallel in conjunctions with qPCR assays.

If you need lot of RNA (ie. lot of qPCR from the same sample) and you want to store it for further measurements, you would prefer the RNA isolation procedures.

However with some cell line the kit dos not work well. For example using the kit with Kasumi1 cell line we could not get proper result, probably due to improper lysis (any suggestions are welcome).

I agree with Gregory prado and Istvan Liko opinion, The Kits is very useful if you have limited sample numbers, its fast , easy to perform , less contamanation , highly sensitve and specific results ,and no high cost than old fashion protocol

For RT-qPCR, I use Promega's GoTaq 2-step and GoTaq 1-step RT-qPCR kits and find that they both work with excellent reproducibility, amplification efficiency, and sensitivity in my experiments using a variety of tissues and primers. I use a separate kit for RNA extraction/isolation which can then be easily tailored to the tissue of interest. It ends up being cheaper to separate the two steps (plus I then have sufficient RNA stocks to repeat experiments as needed) but retains the flexibility to tailor the tissue-sensitive extraction process.

depends of $ and time... but if u need a exceptional procedure and ultra pure extraction continuous whit a exelent qRT-PCR use the protocol of a kit + conventional method (some inserts have a technique conbination).

in other case the kits have more presicion and cuntification procedures estableceds