Total RNA preparation without genomic DNA

A genome and sodium dodecyl sulfate technology, applied in the field of total RNA, can solve the problems of RNA sample degradation, increase the time of RNA extraction, and result uncertainty

Active Publication Date: 2011-12-21
江苏华荣投资发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the DNase required to remove DNA in RNA is very demanding, and RNase-free DNase is required, otherwise the RNA sample will be degraded, so the price is very expensive
DNase digestion also greatly increases RNA extraction time
And during the digestion process, it is difficult to monitor and control the degree of DNA digestion in real time
For different batches of samples, differences in the degree of enzyme digestion may cause uncertainty in the results

Method used

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  • Total RNA preparation without genomic DNA
  • Total RNA preparation without genomic DNA
  • Total RNA preparation without genomic DNA

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Experimental program
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Effect test

Embodiment 1

[0027] a) Streak culture of single colonies of Escherichia coli strains AB1157, AB2463, AB2480, UNC1085 and DH5α on a flat plate, transfer to LB liquid medium, and culture with shaking overnight at 37. Bacteria were harvested by centrifugation. The bacterial cells obtained by centrifuging 1.5 mL of the bacterial liquid were resuspended with 100 μL of a mixed solution of 50 mmol / L glucose and 10 mmol / L sodium oxalate tetraacetate (EDTA).

[0028] b) Add 100 μL of 2% sodium dodecyl sulfate solution by weight, and lyse at room temperature for 3 minutes.

[0029] c) Add 2.5 μL of acidic potassium acetate solution. Mix evenly by inverting, centrifuge at 12000rpm for 10 minutes to remove the precipitate, and take 200μL of supernatant.

[0030] d) Add 120 μL of acidic potassium acetate solution to the supernatant obtained in step c), and 160 μL of a 4.5 mol / L guanidinium thiocyanate stock solution. Add 480 μL of phenol extract, shake and mix. Centrifuge at 10,000rpm for 5 minutes...

Embodiment 2

[0034] Genomic DNA residue detection:

[0035]1 microgram of the total RNA sample obtained in step e), as well as the RNA samples obtained by the acid guanidine thiocyanate-phenol-chloroform (AGPC) method and the SDS-phenol method, were treated with RNase and then amplified with genome-specific primers. According to the comparison with the domain cycle number of the standard sample, it can be seen that the residual genomic DNA of the sample obtained by this method is the least, about 10 per microgram. 3 copies (about 4 pg). Residual genomic DNA in samples obtained by AGPC method and SDS-phenol method is about 10 per microgram respectively. 5 copy and 10 7 copy (see figure 2 ).

Embodiment 3

[0037] Detection of RNA amplification ability:

[0038] Take 1 microgram of the total RNA sample obtained in step e) as a template, and perform reverse transcription using AMV reverse transcriptase. The cDNA obtained by reverse transcription was used as template for semi-quantitative PCR reaction. The primers used are the specific primers of Escherichia coli phr and pgi genes. The pgi gene is a housekeeping gene of Escherichia coli, which encodes phosphoglucose isomerase; the phr gene encodes DNA photorepair enzyme of Escherichia coli. As a result, it can be seen that the phr gene expression level is all lower than the pgi gene in each bacterial strain, and is about about 40%-60% (see image 3 ), the expression levels of the two genes were significantly different, but there was no significant difference in the ratio of each strain. In the control experiment, the genomic DNA of each strain was amplified, which proved the differences in the expression of different genes, and ...

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Abstract

The invention discloses a method for preparing total RNA (Ribonucleic Acid) without genomic DNA (Deoxyribonucleic Acid), which comprises the following steps of: taking lauryl sodium sulfate as a pyrolysis reagent; after cell pyrolysis, carrying out the reaction of potassium acetate and lauryl sodium sulfate to generate lauryl potassium sulfate (PDS) sediment, sedimentating, and simultaneously, commonly sedimentating protein and most of the genomic DNA; absorbing nucleic acid by phenol extraction liquid when guanidine thiocyanate exists, dissociating the absorbed nucleic acid according to molecular weight by potassium acetate with a certain concentration, adding guanidine thiocyanate with a certain concentration and acidity potassium acetate; using phenol to extract and separate the total RNA and the residual genomic DNA; and finally, adopting ethanol to sedimentation so as to obtain a total RNA sample. Compared with the prior art, the total RNA sample has the least residual genomic DNA, and each microgramme sample contains 10<3> copy (about 4pik).

Description

technical field [0001] The invention relates to a method for purifying total RNA without genomic DNA based on phenol extraction. Background technique [0002] With the development of life sciences entering the post-genome era, the study of RNA expression patterns has received more and more attention. Many RNA-related detection methods, such as reverse transcription PCR (RT-PCR), Northern blotting, gene chip and high-throughput RNA sequencing technology are also increasingly widely used. Obtaining high-quality, complete and free total RNA is a prerequisite for the application of RNA research techniques. Currently, there are many methods for extracting RNA, which are mainly classified into two categories. One type is based on phenol extraction. These methods use denaturants to lyse cells and inhibit nucleases, and use phenol extraction to isolate nucleic acids. Commonly used denaturants include guanidinium thiocyanate (GuSCN) and sodium dodecyl sulfate (SDS). Guanidine th...

Claims

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Application Information

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IPC IPC(8): C12N15/10C07H21/02C07H1/06
Inventor 朱国萍徐蕾凌烈锋王鹏曹正宇宋平吕俊靳明明徐雷雷裴云云
Owner 江苏华荣投资发展有限公司
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