Measurement of Cytokine and Chemokine mRNA Using Nonisotopic Multiprobe RNase Protection Assay

互联网2014-02-13

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The multiprobe ribonuclease protection assay (RPA) is a highly sensitive and specific method for simultaneous detection and quantification of several species of mRNA. Three most distinct advantages of the multiprobe RPA method are: high sensitivity and specificity, capacity to simultaneously quantify more than a dozen different mRNA transcripts in a single sample of total RNA, and the flexibility of multiple probes (cDNA, antisense RNA, oligo probe, etc.) that can be used for hybridization with mRNA (Table 1 ). This method allows one to compare relative mRNA levels of multiple genes, or to compare changes in mRNA levels among various treatments or among various tissues. For such comparisons, the results can be normalized based on the amount of mRNA for housekeeping genes.

Table 1 Comparison of Direct RNA Quantification Methods ^a

Method	Sensitivity/features	Advantages	Disadvantages
RPA	High. 1-100 μg RNA/lane Bands correlate with the protected probe size and the amount of hybridized RNA, but not the size of original mRNA.	Measures expression of 10–20 genes in one RNA sample. No need to reprobe. More sensitive than Northern. Tolerates partially degraded RNA. Once the conditions are set up generates more data in less time. Templates and kits available commercially.	Technically demanding.
Northern blot	Low. 10-20 μg RNA/lane. Bands correlate with both the amount and size of of original mRNA.	Traditional gold standard method for measurement of mRNA. Serves both as a qualitative (size of transcript) and quantitative method to evaluate RNA.	Detects one gene per experiment. Needs reprobing for other genes. Signal diminished with reprobing and time. Needs more and higher quality RNA. Large variation from lab to lab.
Dot/slot blot	Intermediate. 4-10 μg RNA/dot or slot. Spots or bands correlate only with the amount but not the size of original mRNA.	One experiment can measure 100 RNA samples. Very small variation in results. Quantification of RNA more accurate than Northern. Needs less experience to run.	Needs large amount of high quality RNA. Laborious.

^a Other (indirect) mRNA quantification methods include reverse-transcriptase polymerase chain reaction (RT-PCR) and Quantikine (R&D Systems) and Xplore (Endogen) colorimetric methods.