miRNA qPCR assay FAQs
1. What is the qSTAR miRNA First-strand cDNA synthesis kit?
qSTAR miRNA First-strand cDNA synthesis kit is for synthesizing miRNA cDNA using Ecoli poly A polymerase and MMLV reverse transcriptase.
2. What are qSTAR miRNA qPCR Primer Pairs and Panels?
qSTAR qPCR Primer Pairs are pre-designed, qPCR tested and ready-to-use primer sets for SYBR Green based qPCR experiments. The primer pairs are also available in a 96-well panel or 384-well panel.
3. Are qSTAR miRNA qPCR Primer Pairs and Panels compatible with cDNAs synthesized by using other manufacturer?s cDNA synthesis kits?
No. The primer pairs are only compatible with the miRNA cDNA synthesis kit provided by OriGene because of the unique linker sequence in the oligo dT.
4. Can qSTAR miRNA qPCR Primer Pairs be used in probe-based qPCR?
No. This kit is only compatible with SYBR Green based qPCR and has not been designed to accommodate any commercial probe-based qPCR.
5. What is the Tm of the primer and what is a typical size of the amplicon?
The Tm of a qSTAR qPCR miRNA Primer is around 55?C, and the amplicon is around 80 bp.
6. My qPCR machine is not on the list of compatible machines on the website. Do you provide miRNA qPCR primer panels in plates compatible with other machines?
Yes, we can provide our primer panels in other plates within approximately one week of receiving the custom order. Please email the catalog numbers of the qPCR primer panels needed plus the catalog number and manufacturer of the plates to firstname.lastname@example.org. Our technical support scientists will prepare a custom quote that can be used to order custom panels using a special TSC1001 code.
7. If I provide a list of miRNA targets I want to assay, can OriGene prepare a custom qPCR primer panel?
Yes, OriGene can design a custom qPCR primer panel. Custom panels are offered for human and mouse miRNA sequences in 10 or 200 reaction-format. Please fill out the custom request form on our website at http://www.origene.com to generate custom-mixed panel of miRNAs.
8. Where can I find the sequence of the primers in each panel?
Each panel is delivered with a USB drive that contains a spreadsheet of primer sequences and locations.
9. How much primer pair is supplied in each well of the miRNA detection panels?
Each well in the 200-reaction format contains 1 nmol of each lyophilized primer (2 nmol total). Please re-dissolve in 200ul of water so the final concentration is 10uM total. Each well in the PCR plate contains 10pmol of the primer pairs. The primers will be re-suspended once the PCR reagents are added to the plate.
10. What is a qPCR template standard?
A sequence specific qPCR template standard is a tube of linear DNA made from a miRNA cDNA cloned in a plasmid. The copy number of a template standard solution is determined by the SYBR Green method and calculation based on MW.
11. Can a template standard be used in a probe-based qPCR?
No. qSTAR miRNA template standards are uniquely designed to function only with OriGene?s qPCR detection system.
12. In your protocol, it is recommended to make six serial dilutions with one log of difference, can I make more dilutions and with different dilution scheme?
Yes, as long as it is diluted in the qPCR detection range and each dilution is mixed thoroughly.
13. Can I use my own buffer to dilute the template standard instead of the buffer in the kit?
Yes, as long as the buffer has no negative effects on qPCR. Use appropriate controls.
14. How should I calculate the copy number of my target of interest?
Since the miRNA cDNA templates in your samples are single-stranded, the actual copy number of your gene of interest is two times the number you would infer in comparison to the qPCR standards. For example, if the copy number of your gene is 5 copy/?l from the standard curve of a qPCR program, the actual number is 10 copy/?l. The reason is that the first cycle for a single-stranded sample is to make the complementary strand; therefore, there is one cycle delay in the PCR reaction compared to the double-stranded cDNA template standard?s reaction.
15. What is the OriGene guarantee on miRNA qPCR primers?
OriGene?s miRNA primers are guaranteed to work in SYBR Green qPCR experiments. In order to determine the functionality of miRNA primers, the U6 control primers from the cDNA synthesis kit must be used along side each amplification experiment. If no U6 transcript is detected by qPCR from your cDNA sample, our scientists will work with you to pinpoint the problem and, if it is determined that our primers are at fault, OriGene will replace them free of charge or credit your account.
16. How should I cite your product?
Here is our guideline:
Full Product Name, OriGene Technologies Inc., Rockville, MD, USA, Catalog #, Lot #.
In addition, we would love to hear from you when the paper is published. Email us a copy of the accepted manuscript and receive a special gift.