[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products ProductsApplicationsServicesResourcesSupport [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[Polymerase Chain Reaction Applications](https://www.sigmaaldrich.com/US/en/applications/genomics/pcr)Reverse Transcription Protocol (One-step Probe Detection) # Reverse Transcription Protocol (One-step Probe Detection) __Section Overview__ - [Reverse Transcription](https://www.sigmaaldrich.com#reverse) - [Equipment](https://www.sigmaaldrich.com#equipment) - [Reagents](https://www.sigmaaldrich.com#reagents) - [Supplies](https://www.sigmaaldrich.com#supplies) - [RT-PCR Method](https://www.sigmaaldrich.com#rt) - [Related Products](https://www.sigmaaldrich.com#products) ## [](https://www.sigmaaldrich.com)Reverse Transcription Reverse transcription (RT) is the process of converting RNA to cDNA using a reverse transcriptase enzyme and dNTPs. The RT step may be performed on total RNA such that a global cDNA is produced that is representative of all of the RNA transcripts in the sample (usually via a two-step protocol), or in a gene-specific approach such that only the RNA of interest is converted to cDNA (usually following a one-step protocol). The following experiments can be used as basic RT protocols that can be modified to suit particular requirements. It is customary to either prepare cDNA using a two-step process with subsequent dilution of the cDNA prior to adding it to the PCR/qPCR, or to prepare a one-step reaction where both processes are carried out sequentially. In some cases it is preferable to measure the target transcript directly, without preparing cDNA from the entire RNA sample. Such situations may include measurements on highly degraded RNA or when there is limiting sample. ## [](https://www.sigmaaldrich.com)Equipment - Quantitative PCR instrument - Laminar flow hood for PCR set up (optional) ## [](https://www.sigmaaldrich.com)Reagents - RNA (Stock, approximately 1 μg/μL) - Quantitative RT-PCR Ready Mix ([QR0200](https://www.sigmaaldrich.com/US/en/product/sigma/qr0200)) - PCR grade water: PCR grade water ([W1754](https://www.sigmaaldrich.com/US/en/product/sigma/w1754) or [W4502](https://www.sigmaaldrich.com/US/en/product/sigma/w4502)) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction. - Forward and reverse primers concentration stocks (10 μM working stocks are suitable for use in single reactions whereas 100 μM working stocks are suitable for use in multiplex reactions). - Specific target detection probes (PCR/qPCR/dPCR Assay Design) concentrated stocks (10 μM working stocks are suitable for use in single reactions whereas 100 μM working stocks are suitable for use in multiplex reactions). - Custom oligos can be ordered at oligos. | | | | |----------------------------------------|-----------------------------------|------------------------------------------| | Instrument | Final Reference Dye Concentration | μL of Reference Dye (per 20 μL Reaction) | | Applied Biosystems 5700 | 1× | 0.2 | | Applied Biosystems 7000 | 1× | 0.2 | | Applied Biosystems 7300 | 1× | 0.2 | | Applied Biosystems 7500 | 0.1× | 0.02 | | Applied Biosystems 7500 Fast | 0.1× | 0.02 | | Applied Biosystems 7700 | 1× | 0.2 | | Applied Biosystems 7900 | 1× | 0.2 | | Applied Biosystems 7900 HT Fast | 1× | 0.2 | | Applied Biosystems 7900HT | 1× | 0.2 | | Applied Biosystems StepOnePlus™ | 1× | 0.2 | | Applied Biosystems StepOne™ | 1× | 0.2 | | Applied Biosystems ViiA 7 | 0.1× | 0.2 | | Bio-Rad CFX384™ | not used | \- | | Bio-Rad CFX96™ | not used | \- | | Bio-Rad MiniOpticon™ | not used | \- | | Bio-Rad/MJ Chromo4™ | not used | \- | | Bio-Rad/MJ Opticon 2 | not used | \- | | Bio-Rad/MJ Opticon™ | not used | \- | | Cepheid SmartCycler® | not used | \- | | Eppendorf Mastercycler® ep realplex | not used | \- | | Eppendorf Mastercycler® ep realplex2 s | not used | \- | | Illumina Eco qPCR | not used | \- | | Qiagen/Corbett Rotor-Gene® 3000 | not used | \- | | Qiagen/Corbett Rotor-Gene® 6000 | not used | \- | | Qiagen/Corbett Rotor-Gene® Q | not used | \- | | Roche LightCycler® 480 | not used | \- | | Stratagene Mx3000P® | 0.1X | 0.02 | | Stratagene Mx3005P™ | 0.1X | 0.02 | | Stratagene Mx4000™ | 0.1X | 0.02 | ## [](https://www.sigmaaldrich.com/US/en/product/sigma/z374903)[](https://www.sigmaaldrich.com)Supplies - Sterile filter [pipette tips](https://www.sigmaaldrich.com/US/en/products/labware/liquid-handling-and-dispensing/pipette-tips) - Sterile 1.5 mL screw-top microcentrifuge tubes ([CLS430909](https://www.sigmaaldrich.com/US/en/product/sigma/cls430909)) - PCR tubes and plates, select one to match desired format: - Individual thin-walled 200 μL PCR tubes ([Z374873](https://www.sigmaaldrich.com/US/en/product/sigma/z374873)) - Plates       - 96-well plates ([Z374903](https://www.sigmaaldrich.com/US/en/product/sigma/z374903))       - 384-well plates ([Z374911](https://www.sigmaaldrich.com/US/en/product/sigma/z374911)) - Plate seals       - ThermalSeal RTS™ Sealing Films ([Z734438](https://www.sigmaaldrich.com/US/en/product/sigma/z734438))       - ThermalSeal RT2RR™ Film ([Z722553](https://www.sigmaaldrich.com/US/en/product/sigma/z722553)) ## [](https://www.sigmaaldrich.com)RT-PCR Method 1. Place kit components and RNA samples on ice. 2. Mix and then centrifuge briefly to collect contents at the bottom of the tube. 3. Refer to __Table 1__ to prepare a master mix that is sufficient to analyze each sample and controls in duplicate plus prepare 10% extra to allow for pipetting error. 4. Refer to __Table 1__ to prepare a master mix that is sufficient to analyze the NO RT enzyme samples and controls in duplicate plus prepare 10% extra to allow for pipetting error, replacing the RT enzyme with PCR grade water. ReagentVolume (μL) per Single 25 μL Reaction Quantitative RT-PCR 2X Buffer, Product No. [QR0200](https://www.sigmaaldrich.com/US/en/product/sigma/qr0200)12.5 Ref dye (optional). Instrument specific0.025 \*Primer F (10 μM)1.125 \*Primer R (10 μM)1.125 \*Probe (10 μM)0.625 PCR grade water8.475 MMLV RT enzyme0.125 Table 1. Reaction Master Mix for One-step Reverse Transcription, Probe Detection. \*The primer and probe concentrations given are suitable for an entry test of the assay but should be adjusted according to the results of optimization. 5. Add 1 μL RNA (250-2,500 ng) to each reaction tube, replacing with water for No Template Controls. 6. Add 24 μL appropriate reaction master mix (from steps 3 and 4) to each well. If using a PCR plate, follow a plate schematic to ensure that the reaction mix, samples and controls are added to the correct wells. 7. After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube. 8. Set the real time qPCR instrument program according to __Table 2__. | | | | |----------------------------------------------|-----------|--------| | Cycling Conditions | Temp (°C) | Time | | First Strand Synthesis | 42-44 | 30 min | | Denaturation/RT inactivation | 94 | 30 sec | | __Steps 1–3 are repeated through 40 cycles__ | | | | Step 1 | 94 | 5 sec | | Step 2 | 55 | 15 sec | | Step 3 | 72 | 10 sec | Table 2.PCR Conditions for One-step RT-qPCR. ## Related Products Sorry, an unexpected error has occurred Response not successful: Received status code 500 __Related Articles__ - [Extract-N-Amp™ Product Guide](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/extract-n-amp-product-guide) - [PCR/qPCR/dPCR Assay Design](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/pcr-qpcr-dpcr-assay-design) - [Cloning Troubleshooting](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/cloning-and-expression/troubleshooting-for-molecular-cloning) - [Extract-N-Amp™ Plant PCR Kits](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/dna-and-rna-purification/extract-n-amp-plant-kits) - [Ultrapure vs. DEPC Treated Water for Molecular Biology Applications](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/dna-and-rna-purification/nuclease-free-water) - [Complete Solutions for PCR Assay Development](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/dna-and-rna-purification/pcr-assay-development) - [Water for Nuclease-sensitive Molecular Biology Studies](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/dna-and-rna-purification/water-for-nuclease-sensitive-molecular-biology-studies) - [Extract-N-Amp™ Tissue Feature](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/dna-and-rna-purification/xnat-feature-article) - [View More](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) __Related Product Categories__ - [Pipette Tips](https://www.sigmaaldrich.com/US/en/products/labware/liquid-handling-and-dispensing/pipette-tips) - [Solvents](https://www.sigmaaldrich.com/US/en/products/analytical-chemistry/analytical-chromatography/solvents) - [NGS library preparation](https://www.sigmaaldrich.com/US/en/products/molecular-biology-and-functional-genomics/ngs-library-prep) - [Laboratory Containers & Storage](https://www.sigmaaldrich.com/US/en/products/labware/sample-handling/laboratory-containers-and-storage) - [Corning®](https://www.sigmaaldrich.com/US/en/products/labware/labware-partners/corning) - [Laboratory Tubes](https://www.sigmaaldrich.com/US/en/products/labware/sample-handling/laboratory-containers-and-storage/laboratory-tubes) - [PCR Supplies & Equipment](https://www.sigmaaldrich.com/US/en/products/labware/pcr-supplies-and-equipment) - [Laboratory sample collection](https://www.sigmaaldrich.com/US/en/products/labware/sample-handling/laboratory-sample-collection) Top __Sign In To Continue__ To continue reading please sign in or create an account. 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