Use of semi-quantitative Northern blot analysis to determine relative quantities of bacterial CRISPR transcripts.
The Northern blot technique is widely used to study RNA. This relatively old method allows one to detect RNA molecules ranging in size from ∼20 to thousands of nucleotides and simultaneously estimate the size of an RNA and detect its degradation/processing products. The method does not rely on enzymes such as reverse transcriptases or RNA ligases used in most advanced RNA detection methods, which can be advantageous since biases in detection of individual RNAs can be avoided. We used this approach to the transcripts of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) phage defense loci in Escherichia coli. CRISPR loci are transcribed into a single long pre-crRNA, which is then processed at multiple sites to generate ∼60 nt fragments (crRNA) each able to mount defense against a specific phage. The Northern blot technique allowed us to estimate the abundance of individual crRNAs and determine stabilities of both pre-crRNA and crRNA. The procedures described in this chapter can be used with very minor modifications to monitor the abundance and stabilities of transcripts of various lengths from many bacterial sources.
Institutes of Gene Biology and Molecular Genetics, Russian Academy of Sciences, Moscow, Russia.
Ksenia
K
Pougach
Konstantin
K
Severinov
D015152:Blotting, Northern / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005557:Formaldehyde / Q000737:chemistry; D055029:Inverted Repeat Sequences / Q000235:genetics*; D007553:Isotope Labeling; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D012322:RNA Precursors / Q000235:genetics / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D022661:RNA, Untranslated / Q000235:genetics / Q000302:isolation & purification
D008567:Membranes, Artificial; D012322:RNA Precursors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D022661:RNA, Untranslated; D005557:Formaldehyde