Whole genome miRNA expression profiling
Accurate high-throughput profiling of miRNAs is a major challenge. Various methods, such as microarrays and bead-based flow cytometry, are available enabling the detection of multiple miRNAs in a single experiment, but such approaches generally require significant amounts of input RNA (>1 µg) and preclude the use of small clinical biopsies or analysis of small subsets of cells or even single cells. In addition, hybridization based detection methods might compromise specific detection of small RNA molecules. Real-time quantitative PCR (RT-qPCR) has superior specificity and sensitivity, down to the single molecule level. Whole genome miRNA RT-qPCR expression profiling requires only minute amounts of input RNA (100 ng of total RNA).
Background
Biogazelle uses a validated miRNA screening pipeline that allows for accurate and sensitive expression analysis of 755 microRNAs by means of real-time quantitative PCR with hydrolysis probe based miRNA assays ( Mestdagh et al., Nucleic Acids Res, 2008). Briefly, 60 ng of total RNA is reverse transcribed using the Megaplex RT stem-loop primer pool (Applied Biosystems), enabling miRNA specific cDNA synthesis of 755 different human miRNAs and small RNA controls. Subsequently, the Megaplex RT product is pre-amplified by means of a 14-cycle PCR reaction with a microRNA specific forward primer and universal reverse primer to increase detection sensitivity. Finally, a 1,600-fold dilution of pre-amplified miRNA cDNA is used as input for a 40-cycle qPCR reaction with miRNA specific hydrolysis probes and primers (Applied Biosystems). All reactions are performed using the gene maximization strategy. The Cq values are subsequently analyzed following an improved version of the global mean normalization procedure described in Mestdagh et al., Genome Biology, 2009. Biogazelle can also provide further biostatistical analysis upon request.
Different steps/processes made by Biogazelle
- RNA sample reception
- Sending of an acknowledgment of receipt
- Quality control with UV-Vis spectrophotometer and an microfluidic electrophoresis system
- Sending of quality control report
- miRNA reverse transcription
- Pre-amplification of cDNA
- qPCR reaction set up
- Measurements in real-time PCR instrument
- Data analysis
- Sending of a full report
List of Cq values
Result table ready for downstream bio-statistical analysis
Samples
For the entire process 100 ng of total RNA is required at a minimum concentration of 10 ng/µl. We also have experience with body fluids like serum, plasma, saliva and sputum.
Good results have been obtained with RNA extracted using Trizol (if well prepared*) or the Qiagen miRNeasy kit.
*Consistent procedure of upper phase take-off (no inhibiting inner phase) and precipitation for all samples.
Pricing
We are extremely flexible to accommodate your needs and we guarantee exceptional value for money. Please contact our service unit (service@biogazelle.com) for more information or a quotation. To provide pricing it is convenient to have an idea about the number of samples you would like to analyze.