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Realtime PCR (MGTP)

Overview

MGTP (multi-gene transcriptional profiling) is a novel form of real-time PCR based gene expression measurement we developed. MGTP measures mRNA copy numbers per cell for unlimited numbers of genes with high precision and reproducibility (1, 2). MGTP has many advantages over conventional real-time PCR:

  1. MGTP uses a master template for high throughput measurement.

  2. MGTP accurately quantifies and normalizes all other genes to 18s RNA.

  3. All MGTP copy numbers are a permanent record of gene expression, and can be directly compared to gene expression abundances in future and past measurements. Fold induction of a single gene across samples, and inter-gene abundance ratios in a multi-gene profile, can be reliably and reproducibly calculated whenever needed even if samples were collected and measured at different times.

Services

  1. Distributing validated primers. We distribute in quantities suitable for 1,000 PCR reactions. Please download  MGTP Primer Library to see available primers.

  2. Distributing total RNA (1 ug) and cDNA (prepared from 100 ng total RNA). We maintain a library of total RNA and cDNA from various tissues and cell lines.

  3. Designing new primers. We design and validate new primers according to our high standards for quantitative real-time PCR. Please provide us the NCBI symbol of a desired gene in your sample submission Excel file.

  4. cDNA preparation for real-time PCR measurement. We prepare cDNA from 100 ng total RNA that is sufficient for 100 real-time PCR reactions (1 ug total RNA in 10 ul RNase free water). For total RNA preparation, we encourage you to use the Qiagen RNeasy kit and to follow our RNA preparation protocol. If you prefer, you are welcome to provide us with cDNA. Please follow our protocol for cDNA preparation. For protocols, please download our Total RNA and cDNA preparation file).

  5. Measuring mRNA copy numbers per cell. We perform MGTP measurements for genes of your choosing from your RNA samples. We will provide mRNA copy numbers per cell for each of your genes and bar charts illustrating the data in our Excel template (see the results worksheets in our sample submission Excel file). We encourage you to obtain duplicate measurements for each gene-sample combination unless you provide us with multiple samples for each experimental setting.

Turnaround and Pricing

Turnaround times are representative and not guaranteed. Pricing for BIDMC and external customers is still being developed.

Sample Preparation

  1. New primer design: Please download our sample submission file and input NCBI symbols in the sample info work sheet. We encourage you to provide us this list at least 2-3 weeks before you submit your samples as it will take at least 14 days to design and validate primers.

  2. RNA preparation: We encourage you to use Qiagen RNeasy kit to prepare RNA samples with on column DNase digest if your samples are derived tissues. Please download our RNA extraction protocol that is based on Qiagen's method.

  3. Sample specification and submission: Please provide your 1 ug RNA samples in microcentrifuge tubes, adding RNase free water to 10 ul. On the lid of each tube, please write an experimental ID consisting of your initials and a unique numerical sample # starting from 101 (see our sample submission form for more details). Store the samples in -800C and deliver samples to RN298 in dry ice.

Scheduling Services

We current receive enquiries only through email. Please contact: Dan Li (dli1@bidmc.harvard.edu) for further details.

Example Analysis

MGTP profile of integrin expression in HUVEC:

References

  1. Shih, S.C., Robinson, G.S., Perruzzi, P., Calvo, A., Desai, K., Green, J., Ali, I., Smith, L.E., and Senger, D. 2002. Molecular Profiling of Angiogenesis Markers. Am J Pathol 161:35-41.

  2. Shih, S.C., and Smith, L.E. 2005. Quantitative multi-gene transcriptional profiling using real-time PCR with a master template. Exp Mol Pathol 79:14-22.

Find a Researcher

Contact Information

Nicole Magner, Administrative Assistant
Center for Vascular Biology Research
Beth Israel Deaconess Medical Center
Research North
99 Brookline Avenue
Boston, MA 02215
617-667-0654
info.cvbr@bidmc.harvard.edu