Metagenomics and Quantitative PCR

By: Chloe McClanahan, Biofiles Edition 6.2

The highly variable regions within the 16S ribosomal RNA (rRNA) gene sequences are being exploited with PCR and metagenomic sequencing to characterize the microbial strains within the gastrointestinal environment. The 16S rRNA sequence is a useful marker: ubiquitous within the microbial population, functionally stable, and large enough to profile against bioinformatic databases. 16S rRNA sequencing is less cumbersome than DNA-DNA hybridization and has been used to ascertain species distribution within the intestinal microbiota. Quantitative PCR can be coupled with 16S rRNA species-specific primers to assess intestinal species population or used solely to determine the effectiveness of pathogenic colonization inhibition by probiotic treatments.

Sigma Life Science offers a broad array of qPCR kits and probes for all detection chemistries and all instrument platforms. Researchers can simply choose between SYBR® Green and probe-based detection, then select from the following formats:

  • Standard qPCR - kits for plate-based and tube-based instruments
  • Capillary tube qPCR - kits for the Roche LightCycler® 2.0 & 1.0
  • High-Throughput qPCR - kits with reference dye premixed into the ReadyMix. Compatible with most ABI™ instruments
  • One-Step qRT-PCR - kits that include reverse transcriptase for streamlined quantification of RNA levels

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SYBR® Green Detection

Standard qPCR

Compatible Platforms: ABI instruments; Roche LightCycler 480; Bio-Rad/ MJ instruments; Stratagene instruments; Corbett Rotor-Gene™ 6000 and Rotor-Gene 3000; Eppendorf Mastercycler® ep realplex instruments

Name Cat. No.
SYBR® Green JumpStart™ Taq ReadyMix™ S4438-20RXN
S4438-100RXN
S4438-500RXN
SYBR® Green JumpStart™ Taq ReadyMix™ S5193-20RXN
S5193-100RXN
S5193-400RXN

Capillary Tube qPCR

Compatible Platforms: Roche LightCycler 2.0 & 1.0

Name Cat. No.
SYBR® Green JumpStart™ Taq ReadyMix™ for Quantitative PCR, Capillary Formulation S1816-100RXN
S1816-400RXN
SYBR® Green JumpStart™ Taq ReadyMix™ S5193-20RXN
S5193-100RXN
S5193-400RXN

High Throughput qPCR

Compatible Platforms: ABI 7300, 7700, 7900, and StepOne ™

Name Cat. No.
SYBR® Green JumpStart™ Taq ReadyMix™ for High Throughput QPCR S9194-20RXN
S9194-400RXN
S9194-2000RXN

One-Step qRT-PCR

Compatible Platforms: ABI instruments; Roche LightCycler 480; Roche LightCycler 2.0 & 1.0; Bio-Rad/MJ instruments; Stratagene instruments; Corbett Rotor-Gene 6000 and Rotor-Gene 3000; Eppendorf Mastercycler ep realplex instruments

Name Usage Cat. No.
SYBR® Green Quantitative RT-PCR Kit 1 kit sufficient for 100 50-μl PCR reactions QR0100-1KT

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Probe-based Detection

Standard qPCR

Compatible Platforms: ABI instruments; Roche LightCycler 480; Bio- Rad/MJ instruments; Stratagene instruments; Corbett Rotor-Gene 6000 and Rotor-Gene 3000; Eppendorf Mastercycler ep realplex instruments

Name Cat. No.
JumpStart™ Taq ReadyMix™ for Quantitative PCR D7440-20RXN
D7440-100RXN
D7440-400RXN
JumpStart™ Taq ReadyMix™ with dUTP D9191-20RXN

High-Throughput qPCR

Compatible Platforms: ABI 7300, 7700, 7900, and StepOne

Name Cat. No.
JumpStart™ Taq ReadyMix™ for High Throughput Quantitative PCR D6442-20RXN
D6442-400RXN
D6442-2000RXN

One-Step qRT-PCR

Compatible Platforms: ABI instruments; Roche LightCycler 480; Roche LightCycler 2.0 & 1.0; Bio-Rad/MJ instruments; Stratagene instruments; Corbett Rotor-Gene 6000 and Rotor-Gene 3000; Eppendorf Mastercycler ep realplex instruments

Name Usage Cat. No.
Quantitative RT-PCR ReadyMix™ 1 kit sufficient for 100 50-μl PCR reactions QR0200-1KT

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PCR Labware

Sigma-Aldrich offers a broad range of Labware optimized for PCR reactions: pipette tips, labtop coolers, films, foils, plates, thermocyclers, tubes, and workstations. The following tables contain a small selection of PCR Labware. For a complete listing of PCR equipment and supplies, visit sigma.com/labware.


Equipment


PCR Tubes


Equipment

Name Cat. No.
Hettich® MIKRO™ 120 Centrifuge Z720089-1EA
Labnet MultiGene™ Gradient Thermal Cycler, with 96 well block Z739472-1EA
Labnet MultiGene™ Gradient Thermal Cycler, with 60 well block Z739693-1EA
Labnet MultiGene™ Mini Thermal Cycler, with 24 x 0.2 ml block Z739928-1EA
Labnet MultiGene™ Mini Thermal Cycler, with 18 x 0.5 ml block Z723436-1EA
Labnet Prism™ R Refrigerated Microcentrifuge Z723878-1EA

PCR Tubes

Name Cat. No.
Agilent Technologies/Stratagene PCR microtubes Z376418-1PAK
Agilent Technologies/Stratagene PCR microtubes Z376426-1PAK
Agilent Technologies/Stratagene PCR microtubes, Caps for 0.2 mL strip tubes Z377910-1PAK
Corning® PCR tubes CLS6531-960EA
Corning® PCR tubes CLS6571-960EA
Corning® PCR tubes CLS6530-1000EA
Corning® Thermowell™ PCR tubes, flat caps, assorted colors CLS3744-1000EA
Corning® Thermowell™ PCR tubes, flat cap, clear CLS3745-1000EA
Corning® Thermowell™ PCR tubes, dome caps, clear CLS3747-1000EA
Corning® Thermowell™ PCR tubes, dome caps, assorted colors CLS3746-1000EA
Corning® Thermowell™ PCR tubes, flat cap, clear CLS3750-1000EA
Corning® Thermowell™ PCR tubes, flat cap, assorted colors CLS3749-1000EA
FlipTube™ microcentrifuge tubes, clear Z738824-500EA
FlipTube™ microcentrifuge tubes, yellow Z738921-500EA
FlipTube™ microcentrifuge tubes, green Z739030-500EA
FlipTube™ microcentrifuge tubes, red Z739154-500EA
FlipTube™ microcentrifuge tubes, blue Z739251-500EA
Greiner™ PCR tubes Z617687-1000EA
Greiner™ PCR tubes Z617717-1000EA
Greiner™ PCR tubes, cap strips Z617709-125EA

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Materials

     
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