Single cell RNA and mutational analysis PCR (SCRM-PCR): a method for simultaneous analysis of DNA and RNA at the single-cell level

一种单细胞、细胞的技术，应用在生物化学设备和方法、微生物的测定/检验等方向，能够解决逆转录酶缺乏校正活性、易出错等问题

Inactive Publication Date: 2017-09-26
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DNA sequences derived from RNA-seq data are often error-prone due to the lack of proofreading activity of reverse transcriptases

Method used

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  • Single cell RNA and mutational analysis PCR (SCRM-PCR): a method for simultaneous analysis of DNA and RNA at the single-cell level
  • Single cell RNA and mutational analysis PCR (SCRM-PCR): a method for simultaneous analysis of DNA and RNA at the single-cell level
  • Single cell RNA and mutational analysis PCR (SCRM-PCR): a method for simultaneous analysis of DNA and RNA at the single-cell level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0166] Example 1 - Primer design and scrmPCR protocol

[0167] The following protocol steps or materials have been implemented in methods from sources already described elsewhere:

[0168] 1) The conditions described in Peixoto et al., specifically:

[0169] a. Reverse transcriptase reaction using reverse-specific primers.

[0170] b. RNA transcript quantification using semi-nested PCR.

[0171] 2) From CellsDirect TM Materials for the one-step qRT-PCR kit (Invitrogen), specifically:

[0172] a. 2X Reaction Mix Cat. No. 11753-100

[0173] b. Included in catalog number 11753-100 III RT / TaqMix

[0174] 3) Conditions described in Protocol PN 100-4109 from Biomark HD System (Fluidigm) (Sanchez-Freire et al.) and http: / / www.fluidigm.com / home / fluidigm / docs / AppNote_2v1Step_pf9.pdf),

[0175] Specifically:

[0176] a. Deposit single cells in 2x reaction mix.

[0177] b. Annealing time for the preamplification step (4 minutes).

[0178] Primer design scheme

[0179] A prim...

Embodiment 2

[0259] Example 2 - Ubiquitous tumor-derived endothelial microemboli in patients with early colorectal cancer

[0260] Patient samples and clinical data. All subjects gave informed written consent to participate. From July 2012 to 2014, according to a protocol approved by the Institutional Review Board (IRB) of the National University of Singapore, Fortis Surgical Hospital, and SingaporeHealth Services (SingHealth) Clinical samples were obtained in April. Serial blood samples from 82 CRC patients were provided by Forth Surgical Hospital (FSH) and National Cancer Center, Singapore (NCC). Blood samples from 45 healthy subjects were provided by the Singapore Consortium of Cohort Studies (SCCS). All samples were collected in EDTA Vacutainer tubes (Becton-Dickinson) and processed within 6 hours at the Institute of Bioengineering and Nanotechnology. Two cases were excluded from analysis due to technical failure of the microfiltration unit. Wherever possible, matched tumor and me...

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Abstract

A method of simultaneously analyzing RNA and DNA in a sample, the method comprising the step (a) contacting the sample with a reverse primer from a first primer pair directed to a target RNA region to effect reverse transcription of RNA into cDNA with a reverse transcriptase; (b) subsequently contacting the sample with (i) a forward primer from the first primer pair directed to a second cDNA region, (ii) a forward and a reverse primers from a second primer pair targeted to a DNA region, and (ii) a DNA polymerase to simultaneously amplify the target cDNA and target DNA region; and (c) analyzing the amplified target cDNA region and / or amplified target DNA region. Also encompassed are uses of the method to analyze gene expression and mutations, kits comprising primers, enzymes, buffers.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority from Singapore Patent Application No. 10201500472R filed on 21 January 2015, the contents of which are hereby incorporated by reference in their entirety for all purposes. technical field [0003] The present invention generally relates to polymerase chain reaction (PCR). In particular, the invention relates to methods for the simultaneous analysis of RNA and DNA. Background technique [0004] The polymerase chain reaction (PCR) technique, developed by Kary Mullis in 1983, provides a method for rapidly amplifying small amounts of specific target DNA. Amplified DNA can be used to facilitate analysis for the presence of DNA sequence variation, mutation, restriction enzyme cleavage, or ligation of oligonucleotide pairs. PCR has become a common and often indispensable tool widely used in medical and biological research laboratories. [0005] Some of these applications incr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2537/143C12Q2539/103C12Q2539/105C12Q2549/119C12Q1/6806C12Q1/6876
Inventor 陈民汉I·西玛
Owner AGENCY FOR SCI TECH & RES
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