Primer extension method RNA analysis
Primer extension method RNA analysis
The primer extension method can be used for RNA mapping and RNA 5' halo. In this method, an excess of a 5' end-labeled single-stranded RNA primer is hybridized to the RNA to be tested, and the primer is subsequently extended with reverse transcriptase to produce a cDNA that is complementary to the RNA template; the length of the cDNA is determined by polyacrylamide gel electrophoresis under denaturing conditions to determine the location of the RNA end, and the amount of cDNA will reflect the level of RNA.
Operation method
Primer extension method RNA analysis
Materials and Instruments
T4 polynucleotide kinase AMV reverse transcriptase yeast tRNA Move I. Materials and equipment Caveat 1) The total halo of the extended reaction products increases proportionally to the K of the annealing time (annealing time not exceeding 12 h). Even increasing the annealing time beyond 12 h does not significantly increase the total amount of the reaction products because the increased portion of the product formation is lost due to non-specific degradation.2) The M of total RNA required is determined by the concentration of the target RNA, and it is best to use pre-tests to determine the RNA concentration paradigm in order to obtain satisfactory results.3) Primers as short as 17 nucleotides have been used successfully, and when setting up primers for the primer extension assay, it is important that the target sequence is located within the 100 nucleotide region at the 5' end of the RNA to minimize the potential for heterogeneous extension products, which can result from the termination or pause of the reverse transcriptase enzyme in the primary structural region of the template KNA. Single-stranded oligonucleotides (unphosphorylated at the 5' end) are a convenient source of primer domains D. In addition, primers can be obtained from the corresponding kernel of the gene by restriction enzyme digestion. Utilizing a pair of oligonucleotides that are 20 to l0 bp apart and can produce bovine . Rarely cleaved sites with different lengths of sticky unterminal are enzymatically cleaved to produce a coding strand and a non-coding strand of different lengths. Radiolabeling of the fragment between the two cleavage sites produces a unique end-labeled fragment, which is visualized on a deteriorated endocannabinoid gel. Electrophoresis and Radiodetection are used to separate it from the non-coding strand. The greater the difference in length between the two strands, the smaller the fragment cut by the restriction enzyme. The better the separation of the probe. A unique end-labeled double-stranded primer can also be used. This is because RNA-DNA heterodimers are more stable than DNA-DNA heterodimers (their optimal hybridization temperature is about 5°C). However, the need to accurately determine the hybridization temperature when using double-stranded primers reduces the efficiency of this technique. Ideally, the primers should be labeled at the 5' end, and the phosphoric acid at the V-terminus of the restriction fragment must be removed by alkaline phosphatase prior to kinase treatment, although 3' labeling is also acceptable if it is used only for quantitative determination of a particular mRNA. The practical application of restriction enzyme-generated probes may be simpler (e.g., it may be important to generate strand labels of different sizes), and the use of homogeneously labeled probes improves the sensitivity of the primer-extension assay. The further the primer is from the cap site, the greater the likelihood that reverse transcriptase action will terminate prematurely at sites of RNA secondary structure or at sites of RMA degradation (e.g., by RNase), and the signal from these discontinuous "premature" bands of the cap site is diminished. If the primer is moved to the 5' end of the secondary structure, which is a strong blocker of elongation, the rate of cap site production can be improved.4) If the labeled primer is first combined with the RNA to be analyzed, the primer and the RNA can be co-precipitated, and the precipitate can be dissolved in DEPC water, so the other components required for the hybridization reaction can be added directly to the newly suspended precipitate, and the primer and the RNA to be analyzed can be mixed with hybridization buffer, and then heated up to 70 ℃ to destroy the secondary structure of the RNA (or the primer), then revert to hybridization.5) Hybridize at a concentration of primer approximately 10 times the molar concentration of the complementary RNA, if this is exceeded too much (especially if the hybridization stringency is below the optimum) it will result in a non-specific initiation of the hybridization. The optimal temperature for hybridization depends on the K of the primer and its base composition. The formula for calculating the unwinding temperature of RNA-DNA hybrid molecules does not apply to the calculation of short-stranded DNA primers. The unlinking temperature for most probes is 45 to 65°C in the indicated buffer. Small-scale experiments should be conducted using temperatures in this range to determine the optimal hybridization temperature for specific primer-mRNA binding & hybridization reactions should be carried out for 3-6 h. RNA is sensitive to heat at high temperatures, so the higher the optimal hybridization temperature, the shorter the recovery time.6) A simple way to test the hybridization efficiency is to perform a modified S1 nucleic caustic acid localization test on the RNA-containing hybrid molecules. This is done by hybridizing oligonucleotides and RNA in the range of hybridization temperatures to be determined for 3-6 h, digesting the residual molecules with S1 nuclease, and then electrophoresing on a denaturing acrylamide gel to determine the amount of protected (hybridized) oligonucleotides. The temperature at which the maximum anti-S1 nuclease marker is produced should be used as the official hybridization reaction temperature.7) The amount of target mRNA should be optimized, i.e. by performing a typical hybridization reaction with different amounts of RNA preparations, in this case with total cytoplasmic RNA, but with poly(A)+poly(A)RNA can be used here, but poly(A) + RNA will give more sensitive and clearer results.8) The ribonuclease inhibitor RNasin can be used in the extension reaction (1ul, approx. 40U), but it is less effective in the hybridization reaction because the secondary structure is eliminated in the holding time and the denaturation reaction at high temperatures denatures can denature the RNasin protein. In addition, the viability of RNasin is strictly dependent on a minimum amount of 1 mmol/L dithiothreitol, and monothiothreitol is also unstable at high plant temperatures. The elongation reaction is performed at 42°C to reduce the formation of secondary structures in the mRNA, which can cause premature termination of reverse transcription. The elongation reaction results in the formation of more than one band in the vicinity of the cap, which may represent the true transcriptional polysubstrate, and it is thought that methylation of nucleotides at or adjacent to the cap site of the mRNA can cause premature termination of the action of the reverse transcriptase in a portion of the molecule. These effects should be taken into account when accurately determining the flood initiation point. The ratio of the several cap-site bands varies between transfected cells of different dumps, but remains constant for a given source of mRNA.9) The cDNA model catalyzed by dove-transferases folds into inches to form additional bands, which is reduced by the addition of actinomycin D to the extension buffer of the tube. Sodium pyrophosphate (80 mmol/L) seems to work better, but it inhibits some sources of reverse transcriptase speculatively. Crossing of primers with endogenous RNA from tissue culture cells or with vector rRNA sometimes results in false bands. This pseudoband can be identified by isolating RNA from cells that do not express the gene for the primer extension reaction and using them as a control. For more product details, please visit Aladdin Scientific website.
10X kinase buffer 5X hybridization buffer solution Tris-HCl MgCl2 DTT actinomycin D dNTP RNasin formamide uptake stain SephadexG-25 NaAc ethanol [r-32P]ATP
Water bath Hybridization oven Electrophoresis equipment
1) Water bath
2) Hybridization oven
3) Electrophoresis equipment
4) T4 polynucleotide kinase, AMV reverse transcriptase
5) 10X kinase buffer: 5 mol/L Tris-HCl (pH 7.6), 0. lmol/LMgCL2, 50 mmol/LDTT, lmmol/L spermine.1 mmol/LEDTA
6) 5X hybridization buffer solution; 2 mmol/LNaCl,50 mmol/LPTPES(pH6.4)
7) 1X extension solution:
lmol/LTris-HCl(pH8.3) 10ul
120 mmol/LMgCl2 10ul
200 mmol/LDTT 10ul
1 mg/ml Actinidione D 5ul
lOmmol/LdNTP 10ul
RNasin 40U 1ul
Add water to I78ul
8) Formamide staining solution: 80% deionized formamide, 45 mmol/L Tris-HCl (pH 8.3), 45 mmol/L boric acid, 1.25 mmol/L LEDTA, 0.02% xylene cyanide.
9) SephadexG-25, resuspend SephadexG-25 with about 50 times volume of TE [10 mmol/LTris-HCl(pH8.0),1.0 mmol/LEDTA], autoclave for 15 min, cool and store at 4℃.
10) Yeast tRNA: Concentration 25 mg/ml, prepared with DEPC water, pay attention to avoid the contamination of RNase, stored in plastic bottles, stored at 20℃.
11) 3mol/LNaAc
12) 70% ethanol: Prepare with DEPC water.
13) [ r-32P ]ATP
II. Methods of Operation
(i) Radiolabeling primers with T4 polyribonucleotide kinase
1) Dissolve the primer in water or TE buffer to a final concentration of 0.4ug/ul, and then add the following components sequentially.
Primer 1 to 5 pmol (0.4ug)
10X kinase buffer 1ul
[ γ-32P ]ATP 35uCi (3000Ci/mmolL)
T4 polyribonucleotide kinase 20U
DEPC water supplement 10ul
2) Mix and incubate at 37℃ for 0.5~lh, add 250 mmol/LEDTA to terminate the reaction.
3) Increase the total volume with TE to 100ul. SephadexG-25 column was used to separate the unlabeled primer from the kinase-primed primer, the first radioactivity peak was collected, and the reaction was incubated at 65℃ for l0 min to inactivate the T4 polynucleotide kinase.
4) Extract with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), add NaAc and MgCl2 to the aqueous phase to a final concentration of 0.3 mol/L and 5 mmol/L, respectively, and add lOugtRNA. 2.5 times the volume of anhydrous ethanol is added. Place on dry ice for 10 min, centrifuge at 12000 g for 30 min at 4°C, and wash the precipitate by centrifugation with 70% ethanol. The precipitate was dried and redissolved in 80ul of sterile water.
(ii) Hybridization
1) In a microfuge tube, mix the following components:
Target RNA 0.1 to 5 fmol (up to 20ug of total RNA)
SX hybridization buffer 4ul
Labeled primer 1~50fmol
Add water to 20ul
2) incubate at 70℃ for 3 min.
3) hybridize at 54℃ for 1.5~4 h
(iii) Extension reaction
1) Add 178ul of extension buffer to the sample plants and place on ice.
2) Add lul of RNasin and 1ul of reverse transcriptase and hold at 42°C for 1h.
3) Add 20ul of RNasin and 2.5 times the volume of anhydrous ethanol, place on dry ice for 10 min, centrifuge at 12000g for 15 min, centrifuge in 70% ethanol and air dry for 2 min.
4) Resuspend the precipitate with formamide staining solution and mix well.
5) Heat denaturation at 90°C for 3 min, then place on ice.
6) Sample 8ul of sample on a denaturing polyacrylamide gel and add DNA molecular mass standard for electrophoresis.