Roche公司的RNase Protection Assay (RPA) protocol

Roche公司的RNase Protection Assay (RPA) Using DIG-Labeled RNA Probes
下载网址： http://www.roche-applied-science.com/PROD_INF/BIOCHEMI/no1_03/PDF/p22_23.pdf

还有一份protocol
http://www.ualberta.ca/PERINATAL/facilities/pdf/RNase%20Protection%20Assay.pdf

可能出现的问题及应对措施
1.No Discrete Bands, Only Smears－－RNA degraded, or the the RNase Digestion was too harsh.Try reducing the concentration of RNase A to 1 mg/ml or digest for a shorter period of time. Check your RNA for condition.

2.Bands Too Large, High Molecular Weight Artifacts－－The RNase Digestion was inefficient and unable to effectively trim down the RNA:RNA duplexes.Try RNasing longer or increasing the RNase concentration. /Incompletely linearized template DNA.

3.Bands in the Negative Control Lane－－ Inefficent RNase Digestion (see above)/Sense template contaminating riboprobe/Insufficent DNase digestion of riboprobe.

4.Many Lower Molecular Weight Bands Under the Main Band－－There can either be premature stop sites in the probe leading to smaller probe sizes, therefore smaller products. This can also stem from overdigestion by RNase A which will break-up the duplexes if the concentration or digestion time is too long.

5.No Signal At All: You did generate an antisense probe right?