I hope this will help you for conducting real time pcr experiment.

I hope this will help you for conducting real time pcr experiment.

It's ok for you to get know about real-time pcr.

Make sure your pirmers have similar efficiencies or a valid comparison will not be possible.

You have to take care of many parameters. It depends on your experiment. What is your template? Then you have to take care of your primers. What dye would you use? It is a SYBR green or a Taqman Assay. If you have any question please let me know. I use qPCR in routine.

Good primer designing give you better result. selection of internal control also important. i think If pcr product size is less than 600 base pairs we can avoid non specific binding of primers.

There is a lot that goes into working out your PCR successfully. But as mentioned before I agree that the most important parameter is good primer designing. You could skim through the attached documents to get a hang of the basic terminologies of a real-time PCR. The actual expertise will only come with experience :). But you could read through similar articles to have a good ground work before starting up.

And here's one more.

Real Time PCR is a machine. which experiment will you design with that?

Primer design: Tm close to 60C with salt adjusted, if possible check the secondary structure formation with mFold

Reaction: You may use only 20 ul reaction volume in place of 50 ul (it works equally good)

For Sybr Green qPCR: 10 μl of Platinum® SYBR Green qPCR SuperMix-UDG (Invitrogen), 0.8 μl (5 μM) of each forward and reverse primer , 1 μl of template DNA and nuclease free water to make up the volume. PCR Cycling: 2 min at 50C and 95C, 40 cycles at 95C for 12 sec, and 60C for 30-60sec

For Taqman qPCR:10 μl of Platinum® Quantitative PCR SuperMix-UDG (Invitrogen), 0.8 μl (5 μM) of each forward and reverse primer, 0.2-0.8 μl (5 μM) probe, 1 μl of template genomic DNA, and X μl of nuclease free water to make up the volume. PCR cycling are as mentioned for Sybr Green qPCR.

Note: use double quencher probe to minimize the background.

These methods are very well validated in our laboratory. Let me know if you have any question regarding anything else.

Cheers!

For detail you may check this paper

Article Development of a Rapid, Sensitive, and Field-Deployable Razo...

for more details. Use my Work book. find on google. kapil jain MTHFR on google. thanks

the MIQE guidelines are ESSENTIAL

http://www.bio-rad.com/evportal/en/AU/LSR/Solutions/LUSO8N4EH/MIQE-and-RDML-Guidelines

http://www.rdml.org/clinchem.2008.112797v1.pdf

RT PCR is a machine used in DNA Quantification to determine the amount of amplifiable DNA.This test reflects both the quality and quantity of the DNA template present in an extracted sample is beneficial to making decisions about how to proceed.Commercial kits for RT PCR ASSAY are available.

what if my amplicon is more than 150bp, how can I decrease the no. of basepairs?

One can use this link to generate primers for RT PCR : https://www.genscript.com/ssl-bin/app/primer. You just have to add DNA sequence and set parameters the way you want and it designs Primers (for e.g. between lenght 50-150bp). In result it wil give different primer set and you can choose the one that you feel suitable. Select those primers set whos Tm value should not vary more than 1 degree C.

Invitrogen/Life Technologies has a free handbook for PDF download:

Real-Time PCR Handbook

http://find.lifetechnologies.com/qpcr/qpcrhandbook/ltfurl-lp/qpcr-handbook-2402CK-4086KY.html

The best references are contained on the MIQE guidelines for sure. Check this "update" by S. Bustin: http://www.biomedcentral.com/1471-2199/11/74

And the site of the initiative. http://www.rdml.org/miqe.php

The editors of many journals have been encouraged to only accept papers that consider these guidelines.

First of all, decide which quantification method you are going to use, Absolute (eg. quantification of pathogen population in a food sample) or Relative (eg. gene expression study between drug-treated and untreated sample) quantification. This will further decide your experimental design. Secondly, choose the chemistry (SYBR Green TaqMan probe, Molecular Beacons, etc). This will be a deciding factor for primer designing, amplicon size and specificity of the experiment. For experimental design, consider and follow the guidelines laid down in MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) (http://www.clinchem.org/content/55/4/611.long). Hope this may be helpful.

I will use relative quantification.

For relative quantification, an internal quantification control (house keeping gene such as GAPDH, 18S rRNA gene) is required. Most of the qPCR reagent comes inclusive of the internal control. I would suggest you to go for TaqMan probe not for SYBR green chemistry. The former is superior to the latter when it comes for the specificity of the assay. You can use designing softwares such as Beacon Designer, Primer Express for designing the TaqMan probes and primers.

Thanks..I'll try to do so.

One suggestion: please consider that the "higher specificity" claimed for TaqMan probes over Sybr chemistry is always relative (and could be much more expensive. some sources of non-specificity are chemistry-independent. Sensitivity is other "advantage" but sometimes is not required or even noticeable.

TaqMan probes can be useful if you multiplex or if specific enough primers cannot be designed. Myself, I have used TaqMan probes when genomic DNA is the target and Sybr when looking at transcripts. However, we prefer to use Sybr chemistry due to ease of use, need for less optimization and lower cost. Most important thing to consider nowadays is the type and number of endogenous references to use in your qRT-PCR experiment (i.e follow transcripts/cDNA). To use of 3 or more endogenous references can be very useful.

One important thing, first optmise your primers that you will use in the experiment in terms of concentration and annealing temperature. Also, try to include all variables that you are comparing in one plate ( one run ) to reduce the bias. Also, If you have insufficient target cDNA, I advice you to run the reaction in 10 microliter instead of 20 to save some target. If you are going to do relative quantification, do not care about the cDNA conc, only it should be fixed for all ur variables

If you are using two separate reactions for your target and reference genes using DNA binding dye, you need to have same amplicon size for both

Dear Himanshi

Firstly you need to decide whether you need to perform absolute or relative quantification. This will depend on your initial question e.g determining HIV viral load within a sample (absolute) or assessing gene expression relative to a reference control (relative).

You set-up will then also largely depend on the machine you have available to you. For examle Roche's LightCycler 480 I/ II will allow for you to practically set up everything on a single plate and perform the relative quantification for you! Another machine will require a little bit more prep work and additional reactions.

The MIQE standards mentioned earlier are an absolute. They will make publication and quite frankly validation much easier!! They speak to selecting the correct reference gene for your tissue sample (there are websites that will aid in this) etc.

It is also important to remember that if you're using a standard curve you need to include your standards, patients and controls all on a single plate in a single run in at least duplicate appearances with negative controls. For cDNA conversion based real-time a genomicDNA control is essential as well.

If you're going to calculate via the 2 delta delta Ct method you need to ensure that you're PCR inefficiencies for each primer pair being compared is relate able - or you results will not be accurate.

The are many other things but I find that those I've mentioned above are what most first-time experiment runners tend to slip up on. They're minor things but can be quite time- consuming and in some cases expensive to repeat once discovered.

Hope this was helpful.

Regards

Nathaniel

Dear Dr. Himanshi

respect all answer mention above

you should know that real time pcr done in many steps:

1- isolation of RNA

this step done by kit

2- convert RNA to cDNA in process reverse transcription

this need kit with certain primer and PCR machine

3- Real time pcr

using specific primer for your target genes and another for your housekeeping gene + prob or Sybergreen + Real time pcr machine

the result here is on computer its the values of Ct

and by certain equation you can calculate

the gene expression

i suggest this ppt for you

Basic of quantitative real time PCR

Presentation Basic of quantitative real time PCR

good luck