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RNA提取（主要内容如下）
- Tips for Handing RNA
- Total RNA Isolation
- mRNA Isolation
- rRNA Isolation
- Others
- Q & A posted in the Method Forum
Basic Procedures for Handing RNA
· (Ambion)
Tips on handing nuclear acid during phenol extraction, ethanol precipitation.
· (Crawford Lab)
· (GSC, WU)
Notes on precipitation of nucleic acids
· (NWFSC)
Resuspending RNA in Formamide has several benefits over storage in water or ethanol. First, formamide will protect RNA from RNases. The RNA is also extremely stable...
· (Eppendorf)
RNA is more susceptible to degradation than DNA. Unlike Deoxyribonucleases (DNases), which require metal ions for activity (and can therefore easily be inactivated with chelating agents such as EDTA), ribonucleases (RNases) have virtually no cofactor requirements and advantage of the 2’ hydroxyl groups adjacent to the phosphodiester linkages in RNA as a reactive species.
· Avoiding Ribonucleases Contamination (NEB)
Why RNA is more susceptible to degradation than DNA and how to avoid it...
Total RNA Isolation
· (Ambion)
Provides very useful information on sample treatment for RNA extraction. Three method are described for preparing tissue lysis.
· (Ambros Lab)
· (Ambros Lab)
· (Tyner Lab)
Detailed protocol for RNA isolation from tissue using lithium chloride method with recipes
· (Ambros Lab)
· (Ambros Lab)
· (Life Technologies)
Isolation of RNA from small quantities of tissue (1 to 10 mg) or Cell (102 to 104) samples using Trizol reagent
· (Crawford Lab)
· (Crawford Lab)
For small amounts of tissue
· (Faulkner-Jones)
Large scale total cellular RNA isolation using 4M guanidinium isothiocyanate lysis buffer and caesium chloride ultracentrifugation from adult organs.
· (Faulkner-Jones)
This method can be used for some adult tissues, most cell lines and embryos. Harvested tissues are homogenised in urea-lithium and the relatively insoluble lithium-RNA salts are precipitated overnight at 4oC. Much of the DNA remains in solution. The recovered lithium-RNA is acid phenol-chloroform extracted to remove contaminating proteins...
· (Life Technologies)
Isolation of Total RNA, Quantitation of Total RNA
mRNA Selection, Storage of Poly A RNA, Formaldehyde Gel Analysis
· Total RNA Isolation (Carl Clark)
This is a guanidine-based method for isolating RNA from zebrafish embryos.
· (Ambion)
Troubleshooting techniques to help overcome problems associated with isolating total RNA from difficult tissues.
· (PMCI Research)
· (PMCI Research)
· - Phenol/SDS method. (Gimila's Lab)
· Total RNA Isolation from Plant (Kay Schneitz Lab)
Detailed protocol for RNA isolation using guanidium thiocyanate with recipes
· Total RNA Isolation from Plant (Petra Klaff)
Detailed protocol for RNA isolation using guanidium thiocyanate with recipes
· (Plant Sciences, Montana Univ)
- De-Paraffinization of Tissue Sections (Arcturus)
Remove paraffin from section for tissue preparation such as nucleic acid extraction.
- RNA Extraction from Paraffin Section (NIH Protocol) (Arcturus)
A Protocol for extracting RNA from paraffin embedded tissue.
mRNA Isolation
· POLY A+ RNA Preparation (Bowtell Lab)
· Poly A+ RNA Preparation (Promega)
· (Breeden Lab)
Prepare mRNA from total RNA using oligo-dT cellulose
· (Life Technologies)
Isolation of Total RNA, Quantitation of Total RNA
mRNA Selection, Storage of Poly A RNA, Formaldehyde Gel Analysis
· (PMCI Research)
· (PMCI Research)
· (PMCI Research)
· (Faulkner-Jones)
Total RNA is first isolated from the tissues or cells and then mRNA is isolated by PolyA+ selection using oligo(dT) cellulose. This is necessary for sll tissue sources rich in RNase (and some cell lines).
· (Faulkner-Jones)
This method can be used to isolate mRNA directly from cultured cells by adding the oligo (dT) cellulose to proteinase K-digested crude cell lysate. Some whole tissues can be lysed directly in the proteinase K solution, whilst others, notably pancreas, need prior extraction of total RNA using guanidinium or lithium-urea. The total RNA can then be PolyA+ selected using oligo(dT) cellulose.
rRNA Isolation
· Phenol Extraction of rRNA (rat liver) (William H. Heidcamp)
This method extracts RNA from rat liver using a phenol extraction which yields predominantly rRNA and tRNA.
Others
· (William H. Heidcamp)
· Ethidium Bromide Spot Test for DNA or RNA Concentration Estimation (Jim Brown Lab)
Quick estimation of DNA and RNA concentration
· Orcinol Determination of RNA (William H. Heidcamp)
· Sucrose Density fractionation of RNA (William H. Heidcamp)
· Determination of Ncleotide Composition of RNA (William H. Heidcamp)
Determine the concentration of each base in RNA sample by UV spectrophotometer
· (Ambion)
When isolating RNA, it helps to have a rough idea of how much total or poly(A) RNA can be recovered from a given amount of tissue or cells.These numbers provide a guideline against which experimental procedures and results can be checked.
- (Beverly Faulkner-Jones)
The best method is the crush and soak method. It can be sued to isolate single-stranded RNA from denaturing gels. The RNA is of high purity but the method is long and inefficient if long transcripts are to be recovered (cf. less than 30% of DNA greater than 3 kb is recovered).
· How to Prepare Molecular Biology grade glycogen (Contributed by Roberto Salvi)
Glycogen can conveniently substitute for tRNA as a carrier for nucleic acid precipitation. Although Molecular Biology grade glycogen can be purchased from a number of vendors, the main disadvantage is that it is very expensive (e.g., About 100 dollars/20-40 mg). Here we present a simple and inexpensive protocol to prepare a large amount of glycogen which is suitable for any kind of Molecular Biology application (we routinely use it for RNA work).
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