Cell Extraction-Free 1-Step RT-qPCR Kit (No ROX)

Description

Intended Use

  • The Master Mix is used for real-time qualitative and quantitative RT-PCR amplifications with SYBR Green dye for extraction-free RNA samples.
  • The master mix is a premixed, 2X concentrated solution that has all the components except for gene-specific primers and RNA template.

Kit Features

  • The master mix contains extra more redundant amounts of the RTase and DNA polymerase, specifically designed to overcome endogenous inhibition in extraction-free RNA samples.
  • For the reverse transcription step, this kit uses a highly efficient Thermophilic Reverse Transcriptase (US patent pending), which is a thermophilic type A polymerase, with optimal temperatures of 60-62°C.
  • The RTase is easily heat-inactivated at ≥90°C for 1min.
  • The RTase efficiently synthesizes a complementary DNA strand on RNA template from a gene-specific primer, ≤1 unit per 20μL of reaction.
  • The RTase reversely-transcribes single digit copies of target RNA molecules consistently.
  • The kit also contains Taq-Fast DNA polymerase which extends more than 300 bases with short cycling program.
  • The concentrations of the primers are variable depending on assay designs and thermocycling protocols (Table 1).
  • The preferable PCR product size is ≤150bp.
  • The kit has three formulations of No ROX, Low ROX or High ROX concentrations for your choice.
    Kit Contents
  • 2X Master Mix (2x1mL/5x1mL/10mL for 200/500/1000 reactions x 20μL) and an instruction for use.
  • The kit also contains Lysis Buffer (2x10mL/1x50mL/1x100mL).

Transportation and Storage

  • The kit can be transported at below 4°C for up to 3 days.
  • The kit should be kept stable in the dark at -20°C for ≤24 months with ≤10 times of freeze-thaw cycles. The kit can be stored at 4°C for a week.

Protocol for extraction-free RNA from cultured cells

  1. Wash 10,000-20,000 adherent cells in a well or suspension cells in a medium with 1X PBS buffer.
  2. Add 50-100ul Lysis Buffer to the well or to the collected cells, and leave for 5min.
  3. Break down the cells through either sonicator, or vigorous vortex for 1 min.
  4. Heat at 95ºC for 10min and then cool down to 4°C.
  5. Up to 4μl of lysed RNA sample can be added to a 20ul final RT-PCR volume.
    Note: the recovery rate of the extraction-free RNA is heavily affected by how completely the cells are broken down.

Setup Reaction and Thermocycling

  1. Thaw 1-Step 2X RT-PCR Master Mix and other reaction components at room temperature, mix each component, centrifuge and then place on ice.
  2. Set up reactions (Table 1).
  3. Seal tubes with flat, optically transparent caps or seal plate with optically transparent film.
  4. Mix and then briefly centrifuge the tubes or plate.
  5. Program PCR instrument with indicated thermo-cycling protocol.
  6. Load PCR tubes or plate and start to run.
  7. Perform data analysis according to the PCR instrument instructions.

Table 1. Setting up a 20μl or 10μl Reaction

Component Volume per 20μl
 Volume per 10μl Final Concentration
2x Mastermix 10μL 5μL 1X
Primersa Variable Variable Each 150-900nM
RNA Templatesb Variable Variable As low as single digit copies of target RNA to ≤1μg total RNA
H2O To 20μL To 10μL

Notes:

  • Each primer’s Tm should be designed ≥60°C, preferably between 62°C to 65°C, using primer3 software for high efficiency and specificity.
  • RNA templates should be extracted by a qualified silica-based kit and eluted with low EDTA TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0-8.3).

Table 2. Standard Thermocycling Protocol

Stage Temperature
 Period Number of Cycles
I 60°C 10min 1
II 95°C 1min 1
III 95°C 10sec 35-40
III 60°C, signal acquisition 60sec 35-40
IV 60°C to 95°C Various 1


Table 3. Three-Step Thermocycling Protocol

Stage Temperature
 Period Number of Cycles
I 60°C 10min 1
II 95°C 1min 1
III 95°C 10sec 35-40
III 60°C 30 sec 35-40
III 68-72°C, signal acquisition 30 sec 35-40
IV 68°C to 95°C Various 1

Notes:

  • The three-step thermocycling protocol in Table 3 increases overall polymerase activity by 50%, a more effective protocol than Table 2.
  • The primer concentration used in Tables 2 and 3 is typically 0.15-0.2uM.

Table 4. Compatible Instruments

qPCR Instrument ROX required by instrument
 Passive dye setup
Bio-Rad® iQ™5, CFX96, CFX384, Opticon, Roche Lightcycler®
Qiagen Rotor-Gene™
Eppendorf Mastercycler®
Cepheid® SmartCycler®		 Not recommended Not necessary
Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™, Agilent Mx™ Low ROX
(50nM final concentration)		 Turn on ROX passive reference dye 
Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ High ROX
(500nM final concentration)		 Turn on ROX passive reference dye 

Precautions

If you order a “No ROX” master mix but you have an Applied Biosystems or Thermo Fisher instrument, please turn off ROX passive reference dye button when setup assays.

Read more

Product form

SKU: CRTQ01-00

Cell Extraction-Free 1-Step RT-qPCR Kit (No ROX)

Intended Use The Master Mix is used for real-time qualitative and quantitative RT-PCR amplifications with SYBR Green dye for extraction-free... Read more

$180.00 Excl. VAT

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      Description

      Intended Use

      • The Master Mix is used for real-time qualitative and quantitative RT-PCR amplifications with SYBR Green dye for extraction-free RNA samples.
      • The master mix is a premixed, 2X concentrated solution that has all the components except for gene-specific primers and RNA template.

      Kit Features

      • The master mix contains extra more redundant amounts of the RTase and DNA polymerase, specifically designed to overcome endogenous inhibition in extraction-free RNA samples.
      • For the reverse transcription step, this kit uses a highly efficient Thermophilic Reverse Transcriptase (US patent pending), which is a thermophilic type A polymerase, with optimal temperatures of 60-62°C.
      • The RTase is easily heat-inactivated at ≥90°C for 1min.
      • The RTase efficiently synthesizes a complementary DNA strand on RNA template from a gene-specific primer, ≤1 unit per 20μL of reaction.
      • The RTase reversely-transcribes single digit copies of target RNA molecules consistently.
      • The kit also contains Taq-Fast DNA polymerase which extends more than 300 bases with short cycling program.
      • The concentrations of the primers are variable depending on assay designs and thermocycling protocols (Table 1).
      • The preferable PCR product size is ≤150bp.
      • The kit has three formulations of No ROX, Low ROX or High ROX concentrations for your choice.
        Kit Contents
      • 2X Master Mix (2x1mL/5x1mL/10mL for 200/500/1000 reactions x 20μL) and an instruction for use.
      • The kit also contains Lysis Buffer (2x10mL/1x50mL/1x100mL).

      Transportation and Storage

      • The kit can be transported at below 4°C for up to 3 days.
      • The kit should be kept stable in the dark at -20°C for ≤24 months with ≤10 times of freeze-thaw cycles. The kit can be stored at 4°C for a week.

      Protocol for extraction-free RNA from cultured cells

      1. Wash 10,000-20,000 adherent cells in a well or suspension cells in a medium with 1X PBS buffer.
      2. Add 50-100ul Lysis Buffer to the well or to the collected cells, and leave for 5min.
      3. Break down the cells through either sonicator, or vigorous vortex for 1 min.
      4. Heat at 95ºC for 10min and then cool down to 4°C.
      5. Up to 4μl of lysed RNA sample can be added to a 20ul final RT-PCR volume.
        Note: the recovery rate of the extraction-free RNA is heavily affected by how completely the cells are broken down.

      Setup Reaction and Thermocycling

      1. Thaw 1-Step 2X RT-PCR Master Mix and other reaction components at room temperature, mix each component, centrifuge and then place on ice.
      2. Set up reactions (Table 1).
      3. Seal tubes with flat, optically transparent caps or seal plate with optically transparent film.
      4. Mix and then briefly centrifuge the tubes or plate.
      5. Program PCR instrument with indicated thermo-cycling protocol.
      6. Load PCR tubes or plate and start to run.
      7. Perform data analysis according to the PCR instrument instructions.

      Table 1. Setting up a 20μl or 10μl Reaction

      Component Volume per 20μl
      		 Volume per 10μl Final Concentration
      2x Mastermix 10μL 5μL 1X
      Primersa Variable Variable Each 150-900nM
      RNA Templatesb Variable Variable As low as single digit copies of target RNA to ≤1μg total RNA
      H2O To 20μL To 10μL

      Notes:

      • Each primer’s Tm should be designed ≥60°C, preferably between 62°C to 65°C, using primer3 software for high efficiency and specificity.
      • RNA templates should be extracted by a qualified silica-based kit and eluted with low EDTA TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0-8.3).

      Table 2. Standard Thermocycling Protocol

      Stage Temperature
      		 Period Number of Cycles
      I 60°C 10min 1
      II 95°C 1min 1
      III 95°C 10sec 35-40
      III 60°C, signal acquisition 60sec 35-40
      IV 60°C to 95°C Various 1


      Table 3. Three-Step Thermocycling Protocol

      Stage Temperature
      		 Period Number of Cycles
      I 60°C 10min 1
      II 95°C 1min 1
      III 95°C 10sec 35-40
      III 60°C 30 sec 35-40
      III 68-72°C, signal acquisition 30 sec 35-40
      IV 68°C to 95°C Various 1

      Notes:

      • The three-step thermocycling protocol in Table 3 increases overall polymerase activity by 50%, a more effective protocol than Table 2.
      • The primer concentration used in Tables 2 and 3 is typically 0.15-0.2uM.

      Table 4. Compatible Instruments

      qPCR Instrument ROX required by instrument
      		 Passive dye setup
      Bio-Rad® iQ™5, CFX96, CFX384, Opticon, Roche Lightcycler®
      Qiagen Rotor-Gene™
      Eppendorf Mastercycler®
      Cepheid® SmartCycler®      		 Not recommended Not necessary
      Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™, Agilent Mx™ Low ROX
      (50nM final concentration)      		 Turn on ROX passive reference dye 
      Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ High ROX
      (500nM final concentration)      		 Turn on ROX passive reference dye 

      Precautions

      If you order a “No ROX” master mix but you have an Applied Biosystems or Thermo Fisher instrument, please turn off ROX passive reference dye button when setup assays.

      Read more

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