mRNA Level Detection

The study of gene expression regulation relies on the identification and quantification of mRNA species encoding specific proteins. mRNA level assays are an important reference for quantifying the effects of RNAi interference . Here, BOC Sciences is committed to providing customers with mRNA quantification methods based on Northern blot analysis, ribonuclease protection assay(RPA) and quantitative reverse transcriptase coupled polymerase chain reaction (RT-PCR) to achieve a comprehensive analysis of the effects of RNAi interference according to your application and testing needs.

What is Northern Blot Analysis?

Northern analysis is a common method for detection and quantification of mRNA levels and offers several advantages over other techniques. Most notably, it is the simplest method for determining transcript size and identifying selectively spliced transcripts and members of multigene families. It can also be used to directly compare the relative abundance of a given message between all samples on a blot. Several ubiquitously expressed mRNA species are used as standards for Northern blots, including β-actin, α-tubulin, β2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and hypoxanthine-guanine phosphoribosyl transferase. Probes specific for 18S or 28S ribosomal RNA (rRNA) may also be used.

Figure 1. Northern blot analysis of apolipoprotein (apo) A-IV and apo A-I mRNA levels in intestine of control (Ct) and apo A-IV transgenic (Tg) mice. (Karen, R.; et al.1998)

What is Ribonuclease Protection Assay?

The Ribonuclease Protection Assay (RPA) works on the same principle as Northern blotting, which is based on the use of antisense probes (radiolabeled or non-isotopic) hybridized to solutions of RNA samples. After incubation for hybridization, the unhybridized probe and sample RNA are enzymatically degraded and the remaining hybrid is electrophoresed through a denaturing polyacrylamide gel and visualized by radioautography or phosphorescence imaging.

Figure 2. Ribonuclease protection assay.

What is Quantitative RT-PCR?

The unparalleled sensitivity of PCR makes RT-PCR the technique of choice for quantifying low-abundance mRNA species and for detecting mRNA in small numbers of cells. RT-PCR can theoretically detect RNA transcripts of any gene, regardless of the scarcity of starting material or the relative abundance of specific mRNAs. Real-time quantitative PCR is extremely accurate, has high throughput, and is less labor-intensive than other available methods.

Figure 3. RT-PCR.

• Quantitative RT-PCR is performed using endogenous standards.

Samples are standardized against endogenous RNA standards to allow comparison of mRNA levels. Endogenous mRNA standards have the advantage of serving as a control for the extent and completeness of RNA recovery, as well as for inter-sample variation in reverse transcription and PCR.

• Quantitative RT-PCR using exogenous standards.

Exogenous RNA standards have the same primer binding sequence as in the target mRNA template, a method also known as competitive RT-PCR. competitive RNA standards are usually prepared by in vitro transcription, and since the target and standard must compete for the same amplification primers, the amount of input target mRNA can be determined based on the standard concentration of equal amplification products from the target and standard. Thus, unlike the co-amplification of endogenous standards, the use of exogenous standards makes absolute quantification possible.

mRNA Level Detection Services from BOC Sciences

Northern analysis provides information about the size of endogenous transcripts and can tolerate multiple probe types. NPA is better suited for multi-target analysis. The high throughput and specificity of RT-PCR makes it more generalizable. The choice of assay depends on factors such as the research question, the type of sample, the desired sensitivity and throughput requirements. Since no single method can provide all the information, a thorough analysis of the effects of RNAi usually requires a combination of techniques. Based on professional and top-notch equipment, BOC Sciences provides you with an improved and optimized assay process based on the above techniques to achieve higher sensitivity and more efficient mRNA quantification to assist you in analyzing the effects of RNA interference.

Please contact us to tell us more about your needs. The expertise of our scientists, engineers and support team are at your disposal!