Single cell mRNA reverse transcription and amplification method

A reverse transcription, single-cell technology, applied in the field of genome sequence analysis and biology, it can solve the problems of low amplification efficiency, biased transcript length, and inability to efficiently transcribe sequences, so as to expand the scope of application and avoid 3' bias. Effect

CN107893100AInactive Publication Date: 2018-04-10XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2018-04-10
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention belongs to the field of transcriptome analysis, and in particular relates to a single cell mRNA reverse transcription and amplification method. 20 to 500 ng of high-quality full-length double-stranded cDNA can be amplified from 1 to 2,000 cells or 10 pg to 20 ng of extracted eukaryotic total RNA as a start by the method within 5-6h. The success rate of reverse transcription and amplification can reach 95% or above, and the amplified full-length cDNA can seamlessly connected with a mainstream sequencing platform. The method is a powerful tool for studying gene expression at the single cell level and greatly expands the application range of RNA-Seq. The cDNA reversely-transcribed and amplified by the method can be used for expression analysis of a very small amount of sample (single cell), early embryonic development studies, tumor cell heterogeneity studies, immune cell population studies, and stem cell differentiation studies.
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Description

technical field

[0001] The invention relates to the field of biotechnology, to the field of genome sequence analysis, and in particular to a method for identifying the breaking point of embryo balanced translocation and the carrying state of balanced translocation. Background technique

[0002] Transcriptome refers to the collection of all transcribed mRNA products in a certain species or a specific cell in a certain physiological function state, including time and space limitations, and is an inevitable link connecting the genetic information of the genome with the proteome of biological functions .

[0003] Transcriptome analysis includes but is not limited to: analysis of coding genes, prediction of translated proteins, splicing of exons and introns, structural analysis and functional analysis of transcripts, secondary structure of mRNA, differential expression of genes, etc. At present, mature and reliable transcriptome analysis methods include RT-qPCR analysis of gene ...

Examples

Embodiment 1

[0061] Example 1 Obtaining single cell (or trace cell) total RNA

[0062] 1. Provide an aliquot of cells growing adherently on a cell culture flask (with medium). In this method, human lymphocyte GM12878 was selected as the material for subsequent experiments.

[0063] 2. Gently pour out the medium in the cell culture flask described in step 1 and discard it. Add 5mL 1x PBS (the following PBS refers to 1×PBS buffer) buffer, wash twice, then pour out the washed liquid gently and discard it. Dilute trypsin with ultrapure water to a concentration of 0.25%, add 3mL into the culture bottle, rotate gently to make the liquid evenly stick to the inner wall of the bottle, and treat at 37°C for 3 minutes. After the incubation, hold the bottle mouth and use the wrist Gently shake the liquid in the bottle with force, suck out the liquid with a pipette gun and place it in a 15mL centrifuge tube for later use.

[0064] 3. Connect the centrifuge tube with the liquid obtained in step 2, pu...

Embodiment 2

[0070] Example 2 reverse transcription to obtain double-stranded full-length cDNA

[0071] 1. Pipette 1.7*N (N is the number of reactions, pay attention to the addition of 10% pipetting loss) μL of reverse transcriptase mixture (component name: RT Enzyme Mix) and add it to the RT Buffer obtained in step 8 of Example 1 , mixed by hand, and centrifuged to form a mixture labeled "RT Mix".

[0072] Wherein said reverse transcriptase mixture contains reverse transcriptase, reverse transcriptase protective agent, BSA, RNase inhibitor.

[0073] The reverse transcription linker is in *GTGAGTGATGGTTGAGGTAGTGTGGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 2, * means blocked by C18 carbon skeleton).

[0074] The template switching oligonucleotide is

[0075] #GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNrGrGLNA-G (SEQ ID NO: 3, # means NH 2 -C6 closed).

[0076] 2. Add 15 μL of "RT Mix" to the cell lysate sample obtained in step 8 of Example 1, place it on ice immediately after centrifugation...

Embodiment 4

[0089] Example 4 Single Cell mRNA Reverse Transcription and Amplified Product (cDNA) Fragment Interruption

[0090] The following are the steps of Covaris M220DNAShearing:

[0091] Power-on inspection

[0092] 1. Check whether the computer fixed on the top of the machine is properly connected to the line of the machine.

[0093] 2. Confirm that the Drip Tray is placed under the machine.

[0094] 3. Insert the operation tube holder (Tube Holder)

[0095] water bath setup

[0096] 1. Pull up the sliding weight (Sliding Weight) on the top of the tube holder (Tube Holder) and rotate it 90°C.

[0097] 2. Use a random water bottle (wash bottle) to add about 15ml of distilled water or deionized water to the center of the bracket. It is advisable that the water level reaches or exceeds the "RUN" marker.

[0098] sample placement

[0099] 1. Pull up the sliding weight (Sliding Weight) on the top of the tube holder (Tube Holder) and rotate it 90°C.

[0100] 2. Put in the sample t...