Master RNA Expression: The Ultimate TriiZol → RT → qPCR Trio Protocol

In molecular biology laboratories, RNA expression profiling is considered the “fundamental of fundamentals”; virtually every workflow—from gene-function validation to disease-biomarker screening—depends on it. Yet many researchers have stumbled: severe RNA degradation, erratic reverse-transcription efficiency, poor qPCR reproducibility… The key to avoiding these pitfalls lies in mastering the “golden trio”: TriiZol lysis-based extraction, reverse transcription to cDNA, and real-time quantitative PCR (qPCR). Here is a bench-ready guide that keeps your RNA-expression studies green-lighted all the way.

 

Stage 1: TriiZol-based RNA extraction—securing the “source water” of your experiment

 

RNA is inherently “fragile”, readily degraded by ribonucleases. TriiZol reagent, with its strong lytic power and RNase-inhibitory properties, is the gold-standard for total RNA isolation. Animal tissue, plant material, or cultured cells—TriiZol handles them all, delivering high-quality RNA.

 

TriiZol (abs60154) Standard PROTOCOL

 

1. Sample preparation

 

Tissue samples:

TriiZol ratio: 1 ml per 50–100 mg tissue; sample volume ≤10 % of TriiZol volume.

(1) Mince tissue; grind under liquid nitrogen or homogenise.

(2) Transfer powder into a 2-ml tube containing 1 ml TriiZol; vortex immediately, keep on ice until all samples are processed.

(3) Lysate should be clear and viscous. For protein-/fat-/polysaccharide-rich tissues (muscle, fat, plant nodules) remove insoluble debris by centrifugation (4 °C, 12 000 × g, 10 min) and collect the supernatant.

 

Cell samples:

(1) Adherent cells: aspirate medium; add 1 ml TriiZol per 10 cm² (one well of a 6-well plate or a 35 mm dish); pipette to ensure complete lysis; transfer to a tube.

Note: Use surface area, not cell number, to determine TriiZol volume; insufficient volume causes gDNA carry-over.

(2) Suspension cells: collect by centrifugation, remove medium completely; add 1 ml TriiZol per 5–10 × 10⁶ animal/yeast cells or 1 × 10⁷ bacterial cells; pipette to homogeneity; transfer to a tube.

Note: Do NOT wash cells before adding TriiZol—washing induces RNase release. Optional: mechanical homogenisation for tough yeast/bacteria.

 

2. Phase separation

(1) Incubate lysate 5 min at RT to dissociate nucleoprotein complexes. (Lysate can be stored at –80 °C at this stage.)

(2) Add 0.2 ml chloroform per 1 ml TriiZol; cap tightly; shake vigorously 15 s; incubate 2–3 min at RT.

(3) Centrifuge 4 °C, 12 000 × g, 15 min. Three phases form: lower orange organic (protein), interphase (DNA), upper colourless aqueous (RNA).

(4) Transfer upper aqueous phase (~60 % of original TriiZol volume) to a fresh tube—avoid the interphase to prevent gDNA contamination.

 

3. RNA precipitation & recovery

(1) Add 0.5 ml isopropanol per 1 ml TriiZol originally used; mix by inversion; incubate 10 min at RT.

(2) Centrifuge 4 °C, 12 000 × g, 10 min; discard supernatant; a gel-like RNA pellet should be visible.

(3) Wash with 1 ml 75 % ethanol (per 1 ml TriiZol used); mix by inversion.

(4) Centrifuge 4 °C, 12 000 × g, 5 min; discard supernatant.

(5) Air-dry pellet 5–10 min at RT or vacuum-dry (do NOT over-dry).

(6) Dissolve in an appropriate volume (e.g. 25 µl) of DEPC-treated water or TE buffer; pipette to resuspend.

(7) Assess concentration, purity (A260/A280) and integrity (agarose gel or Bioanalyzer).

(8) Use immediately or aliquot and store at –80 °C; avoid repeated freeze–thaw.

 

Critical notes:

1. For accurate A260/A280, dilute RNA in quantitation buffer prior to measurement.

2. All consumables and solutions must be RNase-free. Heat-stable plastics: 180 °C, 4 h. Otherwise, soak overnight in 0.01 % DEPC-water, autoclave and dry. Prepare solutions in DEPC-water.

3. Snap-frozen tissue/cells often yield lower RNA quality because freeze-thaw releases endogenous RNases. If extraction cannot be done immediately, homogenise samples in TriiZol and store the lysate at –80 °C.

4. Wear gloves and a mask; avoid talking or breathing over samples.

5. TriiZol contains toxic phenol—use eye protection and gloves; if skin contact occurs, wash with detergent and large volumes of water.

 

Product information

Cat. #	Product	Size	Citations
abs60154	Trizol	100 mL / 500 mL	170 papers

 

Stage 2: Reverse transcription to cDNA—the bridge to qPCR

 

RNA cannot be PCR-amplified directly. Efficient, RNase-free reverse transcription (RT) into stable cDNA governs the accuracy of all downstream quantification. Choosing an optimal RT master mix and strictly controlling reaction conditions are critical.

 

All-in-One 3^rd-Gen RT MasterMix (abs60246) Standard PROTOCOL

 

1. Assemble on ice:

Reagent	Amount
Template RNAa	50 ng–1 µg
All-in-One First-Strand Synthesis MasterMix	4 µl
Nuclease-Free Water	to 20 µl

^a Use high-quality RNA, DNase-treated.

2. Mix gently, spin briefly.

3. Incubate 55 °C 15 min (non-poly-adenylated RNA: pre-incubate 25 °C 10 min).

4. Inactivate at 85 °C 5 min.

5. Place cDNA on ice for immediate use, or store at –20 °C.

 

Technical note:

The master mix contains Oligo(dT)₂₀VN plus random primers; suitable for eukaryotic mRNA, prokaryotic RNA, rRNA, tRNA, but not for miRNA or other small RNAs.

 

Product information

Cat. #	Product	Size
abs60246	All-in-One 3rd-Gen RT MasterMix	100 rxns

 

Alternative: First-strand cDNA Synthesis Mix with gDNA Remover (abs601510) — ready-to-use master mix including genomic DNA elimination. Link: https://www.absin.net/1-cdna/abs601510.html

 

Stage 3: Real-time qPCR—the ultimate weapon for accurate quantification

 

Real-time PCR quantifies cDNA by detecting fluorescence during amplification, providing the gold-standard measure of gene expression. Reliable data require careful attention to reaction setup, thermal profile, and data analysis.

 

SYBR Advanced qPCR SuperMix Kit (abs60087) Standard PROTOCOL

 

I. General considerations

1. Two-step protocol: denaturation 95 °C, combined annealing/extension 60 °C; also compatible with primers T_m < 60 °C.

2. Optimal amplicon length: 60–200 bp.

3. Hot-start activation: 95 °C 2 min.

4. Final reaction volume: 20 µl (96-well) or 10 µl (384-well).

 

II. Reaction setup

1. Thaw 2×SYBR Advanced qPCR SuperMix, template, primers, ROX Reference Dye (optional), and RNase-free water. Prepare master mix according to the table below. Hot-start chemistry allows bench-top assembly—no ice required.

 

Component	96-well (µl)	384-well (µl)	Final conc.
2×SYBR Advanced qPCR SuperMix	10	5	1×
ROX Reference Dye (optional)	2 / 0.1	1 / 0.05	1×
Forward primer	variable	variable	0.7 µM
Reverse primer	variable	variable	0.7 µM
DNA template	variable	variable	≤100 ng/rxn
RNase-Free Water	to 20	to 10	—

2. Mix thoroughly and aliquot into plates/tubes.

3. Add template (≤100 ng) to each well. For two-step RT-qPCR, undiluted cDNA must not exceed 10 % of the final PCR volume.

4. Run the following thermal profile; collect fluorescence during the annealing/extension step.

 

Step	Temp.	Time	Ramp rate	Cycles	Notes
Hot-start	95 °C	2 min	max/fast	—	Polymerase activation
Denaturation	95 °C	5 s	max/fast	35–40a	—
Anneal/Extend	60 °C	10 s	max/fast		Collect fluorescence

 

^a Cycle number depends on template abundance. Always perform melt-curve analysis to verify specificity.

 

Technical specifications

ROX Reference Dye can be pre-added to 2×SYBR Advanced qPCR SuperMix for long-term storage.

2×SYBR Advanced qPCR SuperMix	High-ROX / Low-ROX (ROX volume to add)
1.7 ml	340 µl / 17 µl

High-ROX instruments: ABI Prism 7000/7300/7700/7900HT, StepOne, StepOnePlus, etc.

Low-ROX instruments: ABI Prism 7500/7500 Fast, ViiA7, QuantStudio 3/5/6/7, Stratagene MX series, Chromo4, Opticon, Rotor-Gene 3000, etc.

No-ROX instruments: Thermal Cycler Dice, Rotor-Gene 6000, Bio-Rad CFX, Roche LightCycler 480, Qiagen Rotor-Gene Q, Analytik Jena qTOWER, etc.

 

Product information

Cat. #	Product	Size
abs60087	SYBR Advanced qPCR SuperMix Kit	100 rxns

Success in RNA-expression research relies on the perfect integration of the golden trio: high-quality RNA extracted by TriiZol, efficient reverse transcription, and precise qPCR amplification. Master this workflow and you’ll obtain reliable, reproducible data that accelerate your discoveries.