Protocols_Reverse Transcription (cDNA Synthesis) Protocol - BackmanLab/BackmanLab.github.io GitHub Wiki

Procedure: Reverse Transcription (cDNA Synthesis)

SOP Prepared by: Emily Pujadas Version: 1.0

  1. Scope

The purpose of this SOP is to describe how to obtain cDNA for RT-qPCR experiments.

  1. Description

This protocol aims to describe the procedure of obtaining complementary DNA (cDNA) after a total RNA extraction and quality control (purity/ integrity/ quantification) check. cDNA is a copy of DNA from a single-stranded RNA or messenger RNA (mRNA) template that is made by the enzyme reverse transcriptase. cDNA can be converted to DNA by DNA polymerase. cDNA, like its template RNA or mRNA, contains only certain fragments of the entire genome and can be useful for studying the expression of specific genes.

  1. Personal Protective Equipment
  • Gloves
  1. Potential Hazards + Safety Precautions

  • Nitrile gloves should be used at all times.

  • RNaseZap RNase Decontamination solution should be used to ensure an RNase-free environment. This can be used for cleaning work surfaces, pipettors, and equipment that must be RNase free.

  1. Materials and Reagents
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  1. Equipment

  • Centrifuge (Tissue Culture Room)

  • Thermocycler (Hi-C Room)

  1. Materials

  • RNaseZap RNase Decontamination Solution (*Invitrogen, Cat# AM9780

    -- Stored at RT in Hi-C room)*

  • ReadyScript cDNA Synthesis Mix (Sigma Aldrich, Cat# RDRT -- *Stored

    at -20 C in Tissue Culture Room)*

  • RNA (stock solution ~ 1 ug/uL from total RNA extraction)

  1. Reagents and Solutions
  • RNase-free water
  1. Procedure

Things to do Before Starting:

  1. Place Ready Script cDNA Synthesis Mix on ice

  2. Mix and then centrifuge briefly to collect components at the bottom

    of the tube

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NOTE 1: Perform cDNA synthesis for non-transduced cells as well

Reverse Transcription (cDNA Synthesis):

  1. Determine the number of reactions required, including controls. Calculate the volumes of each component required for all reactions (allow 10% extra for pipetting errors) and combine reagents according to Table P10-26 using 0.2 mL tubes or 96-well plate sitting on ice.

  • If using a PCR plate, follow a plate schematic to ensure that the reagents are added to the correct wells.

  • Controls: no template (water template), reference gene

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  1. After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube

  2. Incubate reaction mix according to Table P10-27.

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  1. After completion of cDNA synthesis, use 1:5 to 1:10 of the first-strand reaction (2-4 ul) for PCR amplification.

Note 2: when using ReadyScript reagents, 2-4 ul undiluted cDNA can be added to PCR without causing inhibition. If desired, cDNA product can be diluted with 10 mM Tris- HCl (pH 8.0), 0.1 mM EDTA and stored at -20 C until further analysis.

Establishment of a Standard Curve:

  1. If necessary, thaw cDNA sample before starting

  2. Pipette 18 uL of nuclease-free water into 5 microfuge tubes and label them 2 through 6

  3. Mix cDNA sample well and pipette 2 uL into tube 2. Pipette up and down to mix.

  4. Using a new tip, pipette 2 uL from tube 2 to tube 3. Pipette up and down to mix. Repeat the same process for tubes 4, 5, and 6.

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  1. Disposals | Spills | Incidents

Waste (see waste segregations and collection SOP for complete information)

  • All waste containers should be closed once ¾ 's and placed into the appropriate location for pick up full.

  • All waste containers should be properly labelled with the following information:

    • PI's name

    • User | Responsible person

    • Content

    • Waste class (e.g. chemical, biohazard, radioactive, etc.)

    • Waste type (e.g. sharps, plastic, glass, metal, etc.)

    • Waste "status" (e.g. liquid, solid, etc.)

[Protocol Specific]{.ul}

  1. Waste

    • Biological samples -- inactivate with 10% bleach for 30 minutes

      and discard in lab sink

  2. Spills | Incidents:

The risk of hazardous spills is minimal to non-existent.

  1. References

https://www.gene-quantification.de/national-measurement-system-qpcr-guide.pdf

SUMMARY FLOWCHART OF PROCEDURES TO CONSIDER IN CASE OF A

xxxx EMERGENCY

[Only when applicable].

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