Preparation of mRNA for Expression Monitoring

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A⁺ ) versus total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.

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Strategic Planning
Basic Protocol 1: Amplification of mRNA for Expression Monitoring and Hybridization to Oligonucleotide Array Chips
Support Protocol 1: In Vitro Transcription of Control Genes and Preparation of Transcript Pools
Alternate Protocol 1: Solid‐Phase Reversible Immobilization (SPRI) Purification of cDNA and IVT Products
Support Protocol 2: Quantitation of cDNA
Reagents and Solutions
Commentary
Figures
Tables

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Materials

Basic Protocol 1: Amplification of mRNA for Expression Monitoring and Hybridization to Oligonucleotide Array Chips

Materials

SuperScript cDNA kit (Life Technologies), including:
5× First Strand Buffer
200 U/µl SuperScript II reverse transcriptase
5× Second Strand Buffer
10 mM dNTPs
10 U/µl E. coli ligase
2 U/µl E. coli RNase H
10 U/µl E. coli DNA polymerase
5 U/µl T4 DNA polymerase
T7T24 primer: 5′‐GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG TTT TTT TTT TTT TTT TTT TTT TTT‐3′ (custom synthesis; HPLC purification is recommended)
RNase inhibitor (Life Technologies or Ambion)
RNase‐free H₂ O (see appendix 2A for DEPC treatment of solutions; prepare from glass‐distilled H₂ O)
Sample RNA: poly(A)⁺ or total RNA
Sense control transcript pool (see protocol 2 )
recipe25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (molecular biology grade; appendix 2A )
7.5 M ammonium acetate
Absolute ethanol
70% (v/v) ethanol in RNase‐free H₂ O, prechilled to −20°C
10× transcription buffer (Ambion)
10× rNTP mix (see recipe )
100 mM dithiothreitol (DTT)
10 mM Bio‐11‐CTP and Bio‐11‐UTP (Enzo Diagnostics)
2500 U/µl T7 RNA polymerase (Epicentre)
RNeasy mini columns with RLT and RPE buffers and collection tubes (Qiagen)
5× fragmentation buffer (see recipe )
recipe20× SSPE (Bio‐Whittaker; also see appendix 2A )
0.5% (v/v) Triton X‐100 (molecular biology grade; Sigma) in RNase‐free H₂ O
10 mg/ml herring sperm DNA (Promega)
500 pM Bio948 (see recipe )
20× antisense control transcript pool (see protocol 2 )
6× SSPET: 6× SSPE containing 0.005% (v/v) Triton X‐100

Thermal cycler (e.g., Perkin‐Elmer 9600 PCR machine with heated lid)
0.1‐ to 10‐µl filtered micropipet tips (Continental)
Lyophilizer
Small, thin‐walled PCR tubes
GeneChip (Affymetrix)
1‐ to 200‐µl filtered gel‐loading micropipet tips (Fisher)
Rotisserie‐type rotator (Appropriate Technical Resources)
50°C oven

Additional reagents and equipment for quantitation of cDNA (see protocol 4 ), for quantitation of DNA by spectrophotometry ( appendix 1K ), and for washing, staining, and scanning the GeneChip (see manufacturer's instructions)

NOTE: Many buffers and enzymes are supplied with the SuperScript II cDNA kit. Kit enzymes that are limiting may be ordered separately from Life Technologies.NOTE: All temperature‐controlled reactions are performed in an appropriate thermal cycler.

Support Protocol 1: In Vitro Transcription of Control Genes and Preparation of Transcript Pools

Plasmids (Table 4.26.1 ; ATCC #87482 to 87490)
2500 U/µl T3 RNA polymerase (Enzo Diagnostics)
25 mM 4rNTP mix: 25 mM each rGTP, rCTP, rATP, and UTP (Ultrapure; Pharmacia Biotech) in RNase‐free H₂ O

Additional reagents and equipment for purifying IVT products (see protocol 3 )

Table 4.6.1 Additional Materials (also see protocol 1 ) Additional MaterialsPreparation of Plasmid Template Controls

Name a	ATCC #	Transcript size (kb)	Sense RNA	Antisense RNA
Linearize with	Polymerize with	Linearize with	Polymerize with
pGIBS‐LYS b	87482	1.0	Not I	T3
pGIBS‐PHE b	87483	1.3	Not I	T3
pGIBS‐THR b	87484	2.0	Not I	T3
pGIBS‐TRP b	87485	2.5	Not I	T3
pGIKS‐BioB	87487	1.1			Xho I	T7
pGIKS‐BioC	87488	0.8			Xho I	T7
pGIKS‐BioD	87489	0.7			Xho I	T7
pGIKS‐CRE	87490	1.0			Xho I	T7

^a Abbreviations: BioB, BioC, and BioD are cloned fragments from the E. coli bioB , bioC , and bioD genes, respectively. LYS, PHE, THR, and TRP are fragments from the Bacillus subtilis lysA, pheA , thrBC , and trpEDCF genes, respectively. CRE is a fragment from the Cre recombinase derived from E. coli bacteriophage P1. PGIBS and pGIKS are derived from the Bluescript KS II vector (Stratagene).

^b pGIBS‐LYS, −PHE, −THR, −TRP, and DAP contain a 40‐nucleotide synthetic poly(A) tract at the 3′ end of the respective genomic fragments derived from B. subtilis . Sense IVT transcripts derived from Not I‐linearized plasmid templates will contain the artificial poly(A) tail. Plasmids linearized with Bam HI prior to T3 IVT will generate sense transcripts without the synthetic poly(A) tract.

Alternate Protocol 1: Solid‐Phase Reversible Immobilization (SPRI) Purification of cDNA and IVT Products

Materials

Carboxy‐coated magnetic beads (PerSeptive BioSystems for cDNA purification; Bangs Laboratories for IVT purification)
recipe0.5 M EDTA ( appendix 2A )
Sample to be purified: cDNA (see protocol 1 , step ) or IVT RNA (see protocol 1 , step , or see protocol 2 , step )
2.5 M NaCl/20% (w/v) PEG 8000 (molecular biology grade; RNase free)
70% (v/v) ethanol in RNase‐free H₂ O
10 mM Tris acetate, pH 7.8 (RNase free)
Magnetic stand (CPG)
Additional reagents and equipment for determining concentration of cDNA (see protocol 4 ) or RNA ( appendix 1K )

Support Protocol 2: Quantitation of cDNA

Materials

PicoGreen dsDNA Quantitation Kit (Molecular Probes), including:
100 ng/µl standard DNA stock solution
20× TE buffer
PicoGreen reagent
cDNA to be quantitated (see protocol 1 and protocol 3 )
Black‐walled 96‐well plate (Corning)
Fluorimager (Molecular Dynamics, model FSI)

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Figures

Figure 4.26.1 Chip analysis overview. Abbreviations: GIT, guanidine isothiocyanate, PE, phycoerythrin; SPRI, solid‐phase reversible immobilization.

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Literature Cited

Literature Cited
	DeAngelis, M.M., Wang, D.G., and Hawkins, T.L. 1995. Solid‐phase reversible immobilization for the isolation of PCR products. Nucl. Acids Res. 23:4742‐4743.
	Lockhart, D.J., Dong, H., Byrne, M.C., Follettie, M.T., Gallo, M.V., Chee, M.S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H., and Brown, E.L. 1996. Expression monitoring by hybridization to high‐density oligonucleotide arrays. Nature Biotechnol. 14:1675‐1680.
	Schena, M., Shalon, D., Davis, R.W., and Brown, P.O. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467‐470.
Key References
	Lockhart et al., 1996. See above.
	This paper established that, in addition to its role as a resequencing tool, the oligonucleotide array could be used as a powerful methodology for transcriptional profiling.
	Schena et al., 1995. See above.
	A seminal paper on the potential of robotics and microfabrication to accelerate cDNA analysis using a highly parallel format.
	Wodicka, L., Dong, H., Mittmann, M., Ho, M.H., and Lockhart, D.J. 1997. Genome‐wide expression monitoring in Saccharomyces cerevisiae. Nature Biotechnol. 15:1359‐1367.
	Demonstration that oligonucleotide arrays can be used to simultaneously monitor expression of all genes of a eukaryotic organism.