Primer extension experiments have some common problems:

1. What is a primer extension experiment?

Primer extension experiments were used to quantify the amount of mRNA and to determine the type of mRNA with low abundance. In addition, primer extension experiments can calibrate the 5'-end of the transcript to determine the precise initiation of transcription. Specific end-labeled primers were annealed to the complementary region of the RNA strand, followed by RNA as a template, reverse transcriptase extension primers to obtain cDNA, and cDNA analysis by denaturing polyacrylamide gel electrophoresis. The length of the cDNA is the number of bases between the labeled nucleotide of the primer and the end of the RNA-5', and the amount of cDNA obtained is proportional to the starting amount of the target RNA. The ideal primer is ssDNA, 20-40 nucleotides in length, complementary to the 5' end region of the transcript to be analyzed. The extension product is less than 150 nucleotides for optimal separation. Primer extension experiments are very sensitive and can detect 2 pg of transcript, which is equivalent to 1 RNA in 1 cell. Primer extension experiments are well suited for quantitative and qualitative analysis of individual genes.

2. What reagents must I use to do such an experiment?

Primer extension experiments require template RNA, either total RNA or poly(A)+ RNA. A specific end-labeled nucleotide or DNA fragment is used as a primer to synthesize cDNA using reverse transcriptase. In addition to RNA templates and labeled primers, reverse transcriptase, AMV or MMLV reverse transcriptase, reaction solutions, deoxynucleotides, polyacrylamide gel reagents, and electrophoresis devices are also required. The results obtained were analyzed by autoradiography and a phosphor imager.

3. What are the components in the primer extension system?

AMV reverse transcriptase and reaction solution in primer extension system, sodium pyrophosphate, positive control RNA template and control primer, PhiX174Hinf I DNA standard molecular reference for dephosphorylation, T4 polynucleotide kinase and solution, sample solution, AMV reversal Enzyme 2X solution 100 mM Tris-HCl (pH 8.3, 42 ° C), 100 mM KCl, 20 mM MgCl 2 , 20 mM DTT, 2 mM each dNTP, 1 mM spermine. The PhiX174 standard molecular reference and T4 polynucleotide kinase were used as primer labels and criteria for determining the length of the extension product. The control RNA gave an extension of 87 bases as a positive control. Additional information on the primer extension system and AMV reverse transcriptase is reviewed in the Technical Manual TB113.

4. What is the purpose of using sodium pyrophosphate in the reaction?

The use of sodium pyrophosphate and spermine in the primer extension reaction increases the yield of the full-length cDNA and inhibits the template RNA from producing a hairpin structure. Although the mode of action was not determined, the result was an increase in the yield of full-length cDNA and inhibition of template RNA to produce a hairpin structure. MMLV is inhibited by sodium pyrophosphate and spermine. If MMLV is used, actinomycin D can be used to increase the yield of full-length cDNA and inhibit the generation of hairpin structure by template RNA. Sodium pyrophosphate and actinomycin D inhibit the synthesis of the second strand of cDNA.

5. What factors need to be optimized for successful primer extension experiments?

The hybridization temperature used in the primer extension experiments is the most critical factor to be optimized. In general, the highest stringency, or the optimum conditions required for complete hybridization of the primer and complementary sequences, is 5 ° C below the primer dissolution temperature. Temperatures below the highest tightness can greatly increase the rate of hybridization. Hybridization should be performed at different tight hybridization temperatures to control hybridization of primers and specific RNA. When the hybridization tightness is increased, the resulting loss of extension product indicates that the primer hybridizes with a related transcript that is not identical. In the presence of a base and a bis-anion, the RNA degrades at a higher temperature, and a formamide hybridization scheme is employed, which effectively lowers the dissolution temperature, thus allowing hybridization to proceed at a lower temperature. The following solutions can be used in the hybridization step: 40 mM PIPES, 1 mM EDTA, 0.4 mM NaCl, 80% formamide. If this hybridization solution is used, the formamide and salt are removed by precipitation with ethanol before the extension step.

6. What is the amount of RNA used for each reaction?

The amount of RNA and primer used per reaction depends on the type of RNA analyzed (total RNA or poly(A) + RNA), the relative abundance of the RNA of interest. If total RNA is used, the starting amount is 10 ug, but the rare signal is analyzed and 100 ug RNA is used for each reaction. Poly(A)+RNA uses 0.1-1.0 ug per reaction, depending on the rich content of poly(A)+RNA in total RNA, and the concentration of specific RNA to be analyzed. The integrity of the RNA of interest needs to be checked prior to primer extension analysis. The total RNA was analyzed by gel and should have intact 18S and 28S ribosomal RNA bands. Northern blot and RT-PCR analysis were used in the primer extension reaction to determine the presence of specific RNA.

7. What is the amount of primer used for each reaction?

RNA was quantified using primer extension experiments and the labeled primer must be more than the RNA of interest. Primers should be 5-10 times more complementary to RNA. However, in general, the amount of specific RNA of interest in total RNA or poly(A)+RNA is unknown. For the unanalyzed transcript, 50 ug total RNA and 100 fmol primers were used for the initial analysis. As all sites in the RNA that are complementary to the primer are saturated, the test signal is proportional to the amount of RNA injected, independent of the concentration of the primer. The primer should be at least 20 nucleotides and the hybridization site should be located 100 bases downstream of the 5'-end of the transcript without self-complementarity. The ssDNA obtained from the restriction fragment can also be used as a probe, and the length should be no longer than 50-100 nucleotides to obtain the best resolution.

8. What are the advantages and disadvantages of using AMV and MMLV?

The primer extension reaction usually uses AMV, which has better performance and is resistant to secondary structures. The extension temperature of the AMV can be up to 55 ° C to eliminate the secondary structure, but at such temperatures, the yield of the primer extension reaction is generally reduced. The optimum temperature for MMLV is 37 ° C. At higher temperatures, MMLV is unstable, but MMLV can synthesize longer products. This feature is not suitable for primer extension reaction. The product obtained by primer extension reaction is generally short. 100-500 nucleotides.

9. What factors can cause non-specific, short and multiple primer extension products?

The degree of difference in the transcripts studied can result in the production of extension products of varying lengths derived from alternative cleavage and the use of different transcription initiation sites. The presence of differential nuclear RNA can also result in the production of multiple extension products. Cross-hybridization of primers and related or unrelated sequences in RNA can also result in the production of non-specific extension products. The product is shorter than predicted to be related to the degradation of the RNA template, or the secondary structure in the transcription product blocks the synthesis of the reverse transcriptase, resulting in a fragmented cDNA. The presence of DNA contamination in RNA samples also resulted in extension products, and actinomycin D (75 ug/ml) inhibited the production of such products. Negative control experiments without RNA template should be performed to determine that there is no RNA and DNA contamination in the reagents used. RNA templates extracted from cells or tissues that do not express the transcript of interest are also used as negative control experiments.

10. How to calculate the amount of RNA converted to cDNA?

The amount of RNA converted to cDNA can be measured for the primer extension product, and the cpm of the no RNA control experiment. The primer extension product and the product in the 'RNA-free control' were separately cut from the gel, and the reading of the gel block was measured in the scintillation fluid to effectively balance the quenching of the polyacrylamide gel and the decay of 32P.

The following example shows how to calculate the conversion of fmol RNA into cDNA. In this example, half of the primer extension product (20 ul/40 ul) was added to the gel for analysis. The primer concentration of the RNA-free control experiment was 100 fmol, and half of the reaction solution was added to the gel for analysis. The reading of the cut tape is:

The sample is 3912 cpm

Primer is 142359 cpm

First calculate the primer cpm / fmol: cpm / fmol = 142359 cpm / [100 fmolX (20 / 40)] = 2847 cpm / fmol

Next, calculate the fmol of the primer extension product, or cDNA: 3912 cpm / [2847 fmol X (20/40)] = 2.75 fmol cDNA. The value obtained indicates the amount of cDNA obtained in the primer extension.

11. What molecular standard reference is used to determine the exact length of the primer extension product?

To determine the exact length of the primer extension product, it should be compared to the isotopically labeled DNA molecule standard reference. The DNA molecule standard reference is labeled at the end, denatured before the denaturing gel, and the primer extension product is also denatured. Molecular standard references of lengths ranging from 20 to 500 nucleotides are useful in determining the length of the primer extension product. The PhiX174Hinf I DNA standard molecular reference for dephosphorylation is very convenient. It has been dephosphorylated and can be labeled as a 5' end. The molecular standard reference is 24-726 nucleotides in length.

12. Can I use primers to separate primer extension products?

The polypropylene gel used for the separation of the primer extension product was 16X18 cm in size and contained 8% polypropylene, 7 M urea, 1' TBE solution. This glue is very similar to the sequencing gel, so sequencing glue is generally available. Care should be taken not to run primer extension products and free primers outside of the gel, especially when quantifying the cDNA.

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