I. Introduction

II. mRNA Detection Kit Procedure

III. Appendix

I. Introduction

A. Product Description

     Real-time RT-PCR is a highly sensitive and reliable method for gene expression analysis for multiple applications, such as the verification of microarray data. Optimal primer design is critical for successful real-time PCR based analysis of gene expression. Carefully designed primers specifically amplify genes of interest, overcoming the challenge of eliminating nonspecific amplification due to the presence of thousands of genes in first-strand cDNA, each potentially available as a PCR template. In addition, primers that provide efficient amplification are important to ensure accurate gene expression results from the commonly used ∆∆CT method, which requires a consistently high degree of amplification efficiency across all experiments. Taking advantage of an experimentally verified, proprietary computer algorithm, BIOTNT has developed high-quality, gene-specific RT-qPCR Primer Assays for gene expression analyses and microarray data validation.

     RT-QPCR Primer Assays are designed for SYBR Green based on real-time PCR detection. The primer design computer algorithm has been developed using an in vitro assay to ensure that the resulting primer sequences generate a single PCR product of the predicted size and a minimal amount of primer–dimer in 30 cycles of PCR amplification. The assay also ensures that the amplification efficiency of the primers is at least 90%. As a result, the algorithm designs highly effective primer sequences for SYBR Green based real-time PCR detection. RT-QPCR Primer Assays are available for every human, mouse, rat, rhesus macaque, fruit fly, and dog gene annotated by the NCBI.

About Primer:

 High performance: Each RT-QPCR Primer Assay is experimentally validated to amplify a single amplicon of the correct size with uniform PCR efficiency. The performance of RT-QPCR Primer Assays for gene expression analysis is as good as or better than that of TaqMan® Assays.

 Complete genome coverage: Primer assays are available for every human, mouse, rat, rhesus macaque, fruit fly, dog, pig, cow, chicken, horse, Zebrafish, rabbit, and Chinese hamster ovary cell gene. The uniform PCR efficiency and PCR conditions of RT² qPCR Primer Assays allow for an easy transition from single gene analysis to multiple gene expression analyses.

 Convenience: With less than 5-minutes of hands-on time, RT-QPCR Primer Assays deliver guaranteed performance when used with BIOTNT optimized instrument-specific RT-QPCR master mixes for QIAGEN/Corbett, ABI, Bio-Rad, Eppendorf, Roche, Stratagene, Takara, and other real-time PCR instruments.

B. Reagents Provided and Storage Conditions

When purchased with RT-QPCR Detection Kit, the kit includes reagents for 500T RT-PCRs; including up to 100 reactions targeting positive control target. Note:that thermostable DNA polymerase is not supplied with the kit. Do not store in a frost-free freezer.

*Store Nuclease-free Water at -20℃、4℃ or room temp.

qPCR Detection Kit is shipped at ambient temperature, But for long time storage, RT-QPCR Detection kit can be used within 12 mouths if the whole kit is stored at -20℃

C. User-Supplied Required Materials

D. Related Products

II. mRNA Detection Kit Procedure

A. Gene Specific QPCR Primer

  The RT-QPCR Detection Kit is the most reliable SYBR® Green-based quantitative real-time PCR assay for gene expression analysis. Our experimentally verified design algorithm yields gene-specific qPCR assays characterized by uniform and high PCR efficiencies and standardized amplification conditions. Every RT-QPCR Primer Assay is subjected to rigorous experimental verification. Single product amplification of the correct size and high PCR efficiency are guaranteed with the appropriate QPCR Master Mixes. The kit includes the specific human VEGF Primers for amplification of the widely expressed VEGF from either the Human Heart Total RNA supplied with the kit or from user-supplied human sample (Figure1).

III. mRNA Detection Kit Procedure

A. Procedure

i. Briefly (10-15 seconds) spin down all reagents.

ii. For each 20µl PCR, mix the following components in a PCR tube.

Note: We suggest adjusting RNA concentration so that 1 μL is used

iii. Quickly centrifuge and place your tubes in your real-time thermal cycler.

iv. Enter and run the appropriate program for your real-time instrument.

C. QPCR system setup

a) Use a two-step cycling program, for the following instrumentation.

Attention: Roche LightCycler 480® Users: Adjust the ramp rate to 1ºC/sec.

b) Use a three-step cycling program, for the following instrumentation

1. The 10-minute step at 95ºC is required to activate the HotStart DNA polymerase.

2. Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle.

3. Different instruments need different lengths of time to detect the fluorescent signal. Choose the appropriate time for the annealing step (55ºC) for your instrument.

Appendix

A. Description of QPCR Primers information

B. Reference:

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2. Xiaoling Tao1，Jinqi Fan1，Guoying Kao1. Angiotensin-(1–7) attenuates angiotensin II-induced signaling associated with activation of a tyrosine phosphatase in Sprague-Dawley rats cardiac fibroblasts[J]. Biology of the Cell, Accepted Article. DOI: 10.1111/boc.201400015

3. Xuelian Chen MD，Yan Liu MD. Topical insulin application improves healing by regulating the wound inflammatory response[J]. Wound Repair and Regeneration,2012,20(3): 425–434. DOI: 10.1111/j.1524-475X.2012.00792.x

4. Jiawen Fan1，Gezhi Xu1,2 . Pharmacologic Induction of Heme Oxygenase-1 Plays a Protective Role in Diabetic Retinopathy in Rats[J].Investigative ophthalmonogy & visual science,2012,(53): 6541-6556. doi: 10.1167/iovs.11-9241

5. Xiangyu Zou1，Guangyuan Zhang1. Microvesicles derived from human Whartons Jelly mesenchymal stromal cells ameliorate renal ischemia-reperfusion injury in rats by suppressing CX3CL1[J]. Stem Cell Research & Therapy,2014,(5):40. doi:10.1186/scrt428