The best kit for what? Realtime PCR is a wide field, so you have to give some more input to get better answers.
Anjan, forr real time PCR of genes, microRNAs etc., I highly recommend TaqMan Expression Assays: http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr/taqman-probe-based-gene-expression-analysis.html
It's maybe a little bit more expensive then other assays, but at the same time TaqMan is a highly selective, stable and reproducible technique. You will save a lot of time!
Good luck
Anjan - Selection of real-time PCR (Q-PCR) reagents or kits will depend on what you want to achieve based on your project. It will also somewhat vary depending on what real-time PCR system (machine) you are using. If you can provide a little detail then it will be easier to give you a best possible suggestion. For SYBR green dye methods - you can use products from Bio-Rad and Applied Biosystems'. For dual labeled probe based Taqman real-time PCR you can use Taqman or 5'-nuclease assay reagents from Bio-Rad and Applied Biosystem’s (now under Thermo Scientific) reagents. A couple of companies sell host and pathway specific panels of genes. Both Bio-Rad and Applied Biosystems sells Taqman assays. The downside is - they are quite expensive. ClonTech/Takara, Promega, Qiagen and Roche also have good real-time PCR reagents and kits. Most of the time SYBR based methods will work fine if the primers are tested to work well with high specificities and efficiencies. Bottom line – you need to give it a try. The other very important aspects with transcriptional profiling are – the quality of RNA you are starting with and cDNA synthesis (RT reactions). Please let me know if you need any further assistance. Good luck.
Deb sir, I will be performing my RT-PCR expt. in Eppendorf RT-PCR system and basically would like to study expreesion pattern of a bacterial gene. so, which kit would serve my target best?
Anjan are you going to quantify the expression profile of only one target gene transcript or a panel of gene’s transcripts? Depending on expression profiling of how many target genes you want to measure your RT reactions and downstream processing will slightly vary. Moreover, you need to select a few housekeeping genes to normalize the expression level of target genes. If you are measuring bacterial transcript levels then you may choose rRNA housekeeping genes (16S rRNA / 23S rRNA) or mRNA housekeeping genes ( rpoB, rpoD, etc.). There are a few other that you may try.
Thanks Deb sir, for your kind and helpful suggestions..! One important thing is regarding use of the method for RT-PCR study Which method would be best.. SYBR-green or the other? Plz suggest! Actually I want to study a gene expression in two different conditions... so which will be better to proceed with?
Detection by SYBR Green is more common. Regarding the choice between PROBE and SYBR:
PROBE
typically done with TaqMan from Life Tech. You need to order specific probes from Life Tech which are sequence specific.
Advantage: more specific
Disadvantage: more expensive. You must order a new Probe for each different target you wish to quantify.
SYBR:
is an intercalating dye that detects anything double stranded
Advantage: most cost effective. don't need to order specific probes for each target
Disadvantage: because SYBR binds to all double-stranded DNA, it may pick up on dsDNA that is not your target.
attached is an application note on gene expression analysis with SYBR Green dye
http://www.kapabiosystems.com/public/pdfs/kapa-sybr-fast-qpcr-kits/KAPA_SYBR_FAST_Gene_Expression_1_Note.pdf
Thank you madam Lauren for your valuable suggestion and the attachment!
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