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MIQE - Minimum Information for Publication of Quantitative Real-Time PCR Experiments

April 20, 2011 18:14 Posted by David Vaudry

An international consortium has published in 2009 the MIQE guidelines to help researchers produce more reliable data. The MIQE guidelines describe the minimum Information that must be provided for publication of Quantitative real-time PCR Experiments. These guidelines should enable other investigators to reproduce the experiments and increase consistency between laboratories. These guidelines will also help reviewers to assess the validity of the protocols used. A list of the MIQE guidelines is presented below :

Checklist of the MIQE guidelines

Importance

Experimental Design

Definition of experimental and control groups

Essential

Number within each group

Essential

Assay carried out by core lab or investigator's lab?

Desirable

Acknowledgement of authors' contributions

Desirable

Sample

Description

Essential

Volume/mass of sample processed

Desirable

Microdissection or macrodissection

Essential

Processing procedure

Essential

If frozen - how and how quickly?

Essential

If fixed - with what, how quickly?

Essential

Sample storage conditions and duration (especially for FFPE samples)

Essential

Nucleic Acid Extraction

Procedure and/or instrumentation

Essential

Name of kit and details of any modifications

Essential

Source of additional reagents used

Desirable

Details of DNase or RNAse treatment

Essential

Contamination assessment (DNA or RNA)

Essential

Nucleic acid quantification

Essential

Instrument and method

Essential

Purity (A260/A280)

Desirable

Yield

Desirable

RNA integrity method/instrument

Essential

RIN/RQI or Cq of 3' and 5' transcripts

Essential

Electrophoresis traces

Desirable

Inhibition testing (Cq dilutions, spike or other)

Essential

Reverse Transcription

Complete reaction conditions

Essential

Amount of RNA and reaction volume

Essential

Priming oligonucleotide (if using GSP) and concentration

Essential

Reverse transcriptase and concentration

Essential

Temperature and time

Essential

Manufacturer of reagents and catalogue numbers

Desirable

Cqs with and without RT

Desirable

Storage conditions of cDNA

Desirable

qPCR Target Information

If multiplex, efficiency and LOD of each assay

Essential

Sequence accession number

Essential

Location of amplicon

Desirable

Amplicon length

Essential

In silico specificity screen (BLAST, etc)

Essential

Pseudogenes, retropseudogenes or other homologs?

Desirable

Sequence alignment

Desirable

Secondary structure analysis of amplicon

Desirable

Location of each primer by exon or intron (if applicable)

Essential

What splice variants are targeted?

Essential

qPCR Oligonucleotides

Primer sequences

Essential

RTPrimerDB Identification Number

Desirable

Probe sequences (or context of the sequence)

Desirable

Location and identity of any modifications

Essential

Manufacturer of oligonucleotides

Desirable

Purification method

Desirable

qPCR Protocol

Complete reaction conditions

Essential

Reaction volume and amount of cDNA/DNA

Essential

Primer, (probe), Mg++ and dNTP concentrations

Essential

Polymerase identity and concentration

Essential

Buffer/kit identity and manufacturer

Essential

Exact chemical constitution of the buffer

Desirable

Additives (SYBR Green I, DMSO, etc.)

Essential

Manufacturer of plates/tubes and catalog number

Desirable

Complete thermocycling parameters

Essential

Reaction setup (manual/robotic)

Desirable

Manufacturer of qPCR instrument

Essential

qPCR Validation

Evidence of optimization (from gradients)

Desirable

Specificity (gel, sequence, melt, or digest)

Essential

For SYBR Green I, Cq of the NTC

Essential

Standard curves with slope and y-intercept

Essential

PCR efficiency calculated from slope

Essential

Confidence interval for PCR efficiency or standard error

Desirable

R2 of standard curve

Essential

Linear dynamic range

Essential

Cq variation at lower limit

Essential

Confidence intervals throughout range

Desirable

Evidence for limit of detection

Essential

If multiplex, efficiency and LOD of each assay

Essential

Data Analysis

qPCR analysis program (source, version)

Essential

Cq method determination

Essential

Outlier identification and disposition

Essential

Results of NTCs

Essential

Justification of number and choice of reference genes

Essential

Description of normalization method

Essential

Number and concordance of biological replicates

Desirable

Number and stage (RT or qPCR) of technical replicates

Essential

Repeatability (intra-assay variation)

Essential

Reproducibility (inter-assay variation, %CV)

Desirable

Power analysis

Desirable

Statistical methods for result significance

Essential

Software (source, version)

Essential

Cq or raw data submission using RDML

Desirable

For more information regarding the MIQE guidelines, click here.

Our experiments meet the recommendations of the MIQE guidelines and all relevant information will be provided to our users together with their data. For any question regarding our procedures, click here to contact us.

What is the integrity of your RNA?

April 19, 2011 18:06 Posted by David Vaudry

Isolation of intact RNA is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise your qRT-PCR results. To verify RNA quality automated capillary-electrophoresis systems are now available. Profiles generated provide key information on RNA concentration and RNA integrity. The Agilent 2100 Bioanalyzer (Agilent Technologies, USA) provides a framework for the control of RNA quality. RNA quality can be quantified with the RNA Integrity Number (RIN) provided by the Bioanalyzer. Samples are evaluated using 6 parameters and a value ranging from 1 to 10 is assigned. The higher the RIN, the better the integrity of your sample. For more information on the impact of RNA degradation on gene expression profiles, click here.

Click here to see The Good, The Bad and The Ugly of RNA quality

Click here for pricing

Click here to contact us for more information

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