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THE ABRF NARG REALTIME PCR SURVEY 2007: TAKING THE PULSE OF THE QUANTITATIVE PCR FIELD

Kevin L. Knudtson1, Pamela S. Adams2, Deborah S. Grove3, Deborah J. Hollingshead4, Timothy C. Hunter5, and Gregory L. Shipley6

NUCLEIC ACIDS RESEARCH GROUP

1University of Iowa, Iowa City, IA, United States, 2Trudeau Institute, Saranac Lake, NY, United States, 3Pennsylvania State University, University Park, PA, United States, 4University of Pittsburgh, Pittsburgh, PA, United States , 5University of Vermont, Burlington, VT, United States, 6UTHSC-Houston, TX, United States

ABSTRACT

INSTRUMENTATION

ASSAY DEVELOPMENT (cont.)

ANALYSIS

Real-time quantitative PCR (qPCR) has evolved to be one of the primary tools used by investigators to specifically detect and quantify nucleic acid levels. In an effort to follow the growth of this technology, the ABRF Nucleic Acids Research Group (NARG) has conducted periodic surveys designed to build a profile of a qPCR facility. The NARG conducted its first qPCR survey in 2004. The current 2007 survey was modeled after the

previous survey. As the current 2007 and illustrate changes in

used.

Participation

such, comparisons will be made between the previous 2004 surveys in an effort to

the in

way the

the qPCR technology has

survey

was

requested

been from

laboratories discussion information laboratories

by posting instructions on qPCR-related electronic groups. The survey was aimed at gathering from academic, pharmaceutical, and commercial that offer realtime PCR technologies as a shared

resource. could also

Individual laboratories that have these technologies participate. A web-based survey was used to collect

information analysis.

on

instrumentation,

chemistries,

staffing,

and

data

METHOD

Scientists from around the world were invited to participate by announcing the survey on the ABRF Discussion forum and the qPCR listserver at Yahoo.com. Submissions were anonymous and monitored only by IP address to eliminate duplicate entries. Participants submitted their answers via a webpage survey form created by phpESP and kindly hosted by Dr. Robert Lyons of University of Michigan. Results were exported

to an Excel spreadsheet and analyzed

NARG.

For

some

questions,

more

by the members of the than one answer was

Realtime Instruments Used by Respondents

0

5

10 15

20

25

30

35

40

ABI 7900HT ABI 7300/7500 Bio-Rad iCycler/MyiQ/iQ5 Stratagene Mx4000/Mx3000P/MX3005P Roche LightCycler ABI 7700

2007 2004

ABI 7000 Bio-Rad (MJ Research) Chromo4/Opticon/Opticon2/MiniOpticon

Cepheid SmartCycler Corbett RotorGene 6000 Roche LightCycler 480 Corbett RotorGene 3000 ABI 5700 Eppendorf RealPlex DNA Technology DT-322 Idaho Technology Rapid Cycler Techne Quantica System

14.4% of respondents use robotics to set up reactions.

The types of manual pipettors used:

    • 66.7

      % single channel

    • 28.8

      % 8 channel

    • 9.2

      % 12 channel.

30% use a repeating pipettor.

Only 5.9 % (9 respondents) use robotics to load the instrument. 8 use the Caliper Twister to load the 7900 and 1 uses the Eppendorf 7075.

ASSAY DEVELOPMENT

Frequency of Use of Different Application Types

Gene expression - Primary validation/quantification

Gene expression - Confirmation of microarray data

Pathogen (viral/bacterial) detection/quantification

Application Type

Allelic discrimination/SNP analysis

Transgene detection/quantification

Biological diversity/contamination

MicroRNA quantification

Methylation detection

45

Respondent Validation Methods (N=148)

Determine PCR efficiency

Assess sensitivity (e.g., dynamic range)

Run melt curve

Met hod

Check for genomic amplification

Sequence amplicon

Run agarose/ polyacrylamide gel

Agilent Bioanalyzer/ BioRad Expirion

Other

0

20

40

60

80

100

120

Number of Respondents

Frequency of Use of Controls/Replicates (N=148)

No Template control (NTC) to check for contamination

Biological replicate (i.e., different sample, same treatment)

Standard curve

Cont rol / Repl i cate

Technical replicate-same cDNA sample following RT reaction

Minus RT (-RT) or RNA control to check for genomic DNA contamination

External (exogeneous) positive control

Technical replicate-same RNA sample prior to RT reaction

External (exogeneous) negative control

Internal Positive control (IPC) to check for PCR inhibition

None/Not applicable

1

2

3

4

Frequency (1=Least, 5=Most)

ASSAYS

Master Mix Used in QPCR Reaction

Commercial 2X SYBRgreen Master Mix

Commercial 2X Master Mix

Homemade Mix

Homemade reaction mix for use with SYBR Green

140

5

Normalizing Genes Used by Respondents

Normalizing Gene

GAPDH b-Actin Other 18SrRNA HPRT Other ribosomal b2Microglobulin TBP GUS Cyclophilin N/A- None ABL Ubiquitin Tubulins 28SrRNA 16SrRNA YWHAZ S100A8 PGK1 AnnexinA2

2004 2007

0

10

20

30 40 Number of Respondents

50

60

Number of Normalization Genes Used (N=116)

Quantification Standards Used By Respondents

4 (3)

5 or more (1)

3 (16)

2 (17)

1 (79)

~70% 0f Respondents Evaluate Only 1 Normalizing Gene

Standard

Plasmid, linear

PCR product

Pooled cDNA

Genomic DNA

Commercial RNA

In vitro Trans RNA

None

Oligonucleotide

Plasmid, not linear

Other

Bacteria

Plasma

Plasmid + Stds

  • 0

    10

20

30

Number of Respondents

40

Instrument Post-Run Settings Used For Analysis

Respondent Data Analysis Methods

P o st-R u n Settin g s

Auto Ct/Auto BL

Manual Ct/Auto BL

Manual Ct/Manual BL

NA

Other

Analysis Method

Std Curve ddCt REST/REST-XL LineRegPCR qBase DART-PCR Q-Gene Other Stratagene GeNorm GenEx NA Roche Ct or dCt Internal Std

  • 0

    10

20

30

40

50

60

Number of Respondents

70

80

90

  • 0

    20

40

60 80 Number of Respondents

100

70

50

120

permissible. A sampling of the results are presented poster. This poster and complete results (raw data) made available on the ABRF NARG web page.

in this will be

DEMOGRAPHICS & FACILITY

Respondent Years of Experience

2 77

3

53

1

11

Number of Respondents

50

45

40

35

30

25

20

15

10

2007 Manager 2007 Staff 2004 Responder

5

5

0

<1

1-2

2-3

3-4

Years

4-5

>5

No Manager or No Staff

Institution Type

Respondent Lab Type

Government (25)

120

Private Research Foundation (11)

100

2007 2004

Number of Respondents

80

60

40

20

Academic (Univ/Hospital) (88)

Commercial/ Industrial (26)

Not a Core

Core Lab With Other Services

Core Doing qPCR Only

Lab Type

Zygosity testing

ChIP Assays

1

Primer/Probe Design Software Used (N=147)

RealTimeDesign (Biosearch Technologies) (10)

Roche Universal Probe Library (5)

Other (16)

Vector NTI (Invitrogen) (11)

Primer Express (ABI) (77)

SciTools (Integrated DNA Technologies) (10)

LightCycler Probe Design (7)

Oligo (MBI) (8)

Beacon Designer (Premier Biosoft) (24)

Primer 3 (MIT) (62)

Reporter Dyes Used (N=149)

SYBR dyes (85)

Yakima Yellow (3)

FAM (122)

Texas Red (8)

ROX (28)

Other (10)

CAL Fluor Orange/Red (5)

VIC (47)

JOE (30) HEX (37)

TAMRA (20)

TET (15) CY5 (22)

CY3 (9)

2

3

4

Frequency (1=Least, 5=Most)

Frequency of Performing Multiplex Reaction (N=150)

Always (10)

Never (56)

Most of the time (18)

Sometimes (32)

Rarely (34)

Quencher Dyes Used (N=136)

Not applicable (22)

TAMRA (68)

MGB non- fluorescent quencher (47)

DABCYL (7)

Iowa Black (3)

BHQ-1,2,3 (66)

5

Homemade Reaction mix for use with probes

Commercial Core PCR Reagent Kit

Commercial SYBRgreen Core PCR Reagent Kit

Commercial SYBR Green Master Mix for FAST cycling protocols

Commercial Master Mix for FAST cycling protocols

0

10

20

3%

>10-mer

Don't know

2%

Random Primer Length Used

Decamers

5%

Nonamers 8%

Octamers 8%

Hexamers 74%

RNA Purification Methods

Column-/matrix-based

Phenol/column-based

Magnetic bead-based

Detergent-based

2007 2004

0

20 40 60 80 Number of Respondents

100

30

2007 2004

40 50 60 Number of Respondents

70

80

90

100

DNase Treatment

Alw ays

Sometimes

Never

Sample is provided

2007 2004

0

10

20 30 40 50 Number of Respondents

60

70

Reverse Transcription Primer Used

Random primers

Oligo(dT)

Gene-specific primer

Random primers and oligo(dT) mixed

Sample is provided

2007 2004

0

10 20 30 40 50 Number of Respondents

60

SUMMARY

The Average Respondent

  • Works in academia in North America and not in a core facility

  • Runs 0-1000 wells/month and has less than 2 years of

experience and does not use robotics

  • Measures gene expression using SYBRgreen ® or Taqman

  • Designs his/her own assays

  • Validates his/her assays by determining PCR efficiency

  • Runs biological replicates and NTC controls

  • Uses RNA as a template that was purified using a column

  • Uses two step reactions for transcript analysis

  • Transcribes his/her RNA with a MMLV RT at 420C using oligo

dT or random primers

  • Uses a heat-activated Taq and a commercial master mix

  • ROX as a reference dye in a 25 μl reaction

  • Analyzes his/her data using a standard curve obtained from a

plasmid, PCR product, transcribed RNA, or genomic DNA

  • Normalizes to GAPDH, β-actin or 18S rRNA

  • Validates PCR efficiency in every assay, considering an

efficiency of >90% to be acceptable

60

140

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