Complete Guide to RNA Expression Research: Step-by-Step Manual for the TriiZol, Reverse Transcription and qPCR Triple Kit

In molecular biology laboratories, RNA expression analysis is regarded as the "fundamental foundation". It is almost indispensable for gene function verification and disease biomarker screening. However, many researchers have encountered experimental pitfalls: severe RNA degradation after isolation, variable reverse transcription efficiency, poor repeatability of quantitative fluorescence detection results, and so on. In fact, the key to solving these problems lies in the "three core kits": TriiZol lysis & RNA extraction, reverse transcription for cDNA synthesis, and quantitative real-time PCR (qPCR) detection. Today, we will elaborate on the core operating essentials of this golden workflow to streamline your RNA expression research!

 

Stage 1: RNA Isolation via TriiZol Method — Secure the Source of Valid Experimental Samples

 

RNA is inherently labile and susceptible to degradation by ribonucleases. With potent lysis capacity and robust RNase inhibitory activity, TriiZol reagent serves as the gold standard for total RNA extraction. It works efficiently with animal tissues, plant specimens and cultured cells to yield high-integrity RNA.

 

Standard Operation PROTOCOL for TriiZol(abs60154)

 

1. Sample Pretreatment

 

Tissue Specimens:

TriiZol dosage: Add 1 mL TriiZol per 50–100 mg tissue, and the sample volume must not exceed 10% of the TriiZol volume.

(1) Cut animal or plant tissues into small fragments, grind in liquid nitrogen or homogenize with a tissue homogenizer.

(2) Rapidly transfer ground tissue powder into a 2 mL microcentrifuge tube preloaded with 1 mL TriiZol, mix thoroughly via vortexing on ice until all samples are processed.

(3) The lysate should appear clear, transparent and viscous. For protein-, lipid- or polysaccharide-rich tissues (e.g., muscle, adipose tissue, plant nodules) containing insoluble debris post-homogenization, centrifuge at 12,000 × g for 10 min at 4 ℃, then transfer the supernatant to a new microcentrifuge tube.

 

 Cell Samples:

(1) Adherent cells: Aspirate complete culture medium completely, add 1 mL TriiZol per 10 cm² culture area (single well of 6-well plate or 35 mm culture dish). Pipette up and down repeatedly to ensure full cell lysis, then transfer lysate to a microcentrifuge tube.

Note: TriiZol dosage is determined by culture dish surface area rather than cell count; insufficient TriiZol may lead to genomic DNA contamination in isolated RNA.

(2) Suspension cells: Harvest cells by centrifugation and remove all supernatant. Add 1 mL TriiZol per 5–10 × 10⁶ animal/plant/yeast cells or per 1 × 10⁷ bacterial cells. Pipette repeatedly to achieve complete lysis and transfer to a microcentrifuge tube.

 Note: Do not wash cells prior to TriiZol addition to prevent RNA degradation. Homogenizers may be used for lysis of certain bacterial or yeast strains when necessary.

 

2. Phase Separation

(1) Incubate lysate at room temperature for 5 min to fully dissociate nucleic acid-protein complexes. (Note: Samples can be stored long-term at -80 ℃ at this stage.)

(2) Add 0.2 mL chloroform per 1 mL TriiZol, tightly cap tubes, shake vigorously for 15 s, and incubate at room temperature for 2–3 min.

(3) Centrifuge at 12,000 × g for 15 min at 4 ℃; samples will separate into three phases: orange lower organic phase (protein), interphase (genomic DNA), and colorless upper aqueous phase.

(4) Transfer the upper aqueous phase containing total RNA to a new microcentrifuge tube; the aqueous phase volume accounts for 60% of the initial TriiZol volume used.

Note: Avoid touching the interphase during aqueous phase transfer to prevent genomic DNA contamination

 

3. RNA Precipitation & Recovery

(1) Add 0.5 mL isopropanol corresponding to the initial 1 mL TriiZol volume, invert tubes several times for mixing, and incubate at room temperature for 10 min.

(2) Centrifuge at 12,000 × g for 10 min at 4 ℃, discard supernatant; a gelatinous RNA pellet will be visible.

(3) Add 1 mL 75% ethanol per initial 1 mL TriiZol volume, invert repeatedly to wash the pellet.

(4) Centrifuge at 12,000 × g for 5 min at 4 ℃ and discard supernatant.

(5) Air-dry tubes upside down for 5–10 min at room temperature or vacuum-dry briefly (avoid vacuum centrifugation, as over-dried RNA becomes hard to resuspend).

(6) Resuspend RNA pellet in an appropriate volume (e.g., 25 μL) of DEPC-treated ddH₂O or TE buffer via repeated pipetting.

(7) Quantify RNA concentration, purity and integrity using agarose RNA electrophoresis and ultraviolet spectrophotometry.

(8) Purified RNA should be used immediately or aliquoted and stored at -80 ℃ to avoid repeated freeze-thaw cycles.

 

Precautions:

1. To measure A260/A280 ratio of RNA, dilute samples with RNA quantification buffer prior to detection.

2. All microcentrifuge tubes, pipette tips and related buffers must be RNase-free. Heat-resistant labware can be baked at 180 ℃ for 4 h to eliminate RNases; other consumables can be soaked in 0.01% DEPC water overnight, autoclaved and dried. Buffers must be prepared with DEPC water: add 0.01% DEPC to double-distilled or Milli-Q water, incubate overnight, then autoclave to obtain DEPC-treated water.

3. Total RNA extraction from frozen cells or tissues generally yields lower-quality RNA than fresh specimens, as intracellular RNases are released and degrade nucleic acids during freeze-thawing. If RNA extraction cannot be performed promptly, lyse samples with TriiZol first before freezing for storage.

4. Wear disposable nitrile gloves during all operations; refrain from exhaling or speaking toward RNA samples to avoid RNase contamination. Disposable face masks are recommended.

5. TriiZol contains toxic phenol; prevent skin contact and inhalation. Wear protective goggles or transparent face shields to avoid splashes into eyes. In case of skin exposure, rinse thoroughly with abundant detergent and water; seek medical advice if irritation persists.

6. For personal safety, wear lab coats, gloves and other protective equipment before handling reagents.

 

Product Information

Catalog No.	Product Name	Specifications	Cited References
abs60154	TriiZol Reagent	100mL/500mL	170 publications

 

Stage 2: Reverse Transcription for cDNA Synthesis — Construct the Bridge Between RNA and qPCR

 

RNA templates cannot be directly amplified via PCR. Reverse transcription generates stable complementary DNA (cDNA) from RNA templates, and the efficiency of this step directly determines the accuracy of subsequent quantitative detection results. Selection of appropriate reverse transcription master mix and strict control of reaction conditions are critical for experimental success.

 

Standard Operation PROTOCOL for All-in-One 3rd-Gen First-Strand Synthesis Master Mix(abs60246)

 

1. Prepare the following reaction system on ice: 

Reagent	Volume
Template RNA a	50 ng~1 μ g
All-in-One First-Strand Synthesis MasterMix	4 μ l
Nuclease-Free Water	To 20 μ l

a. High-quality RNA templates pre-treated to remove genomic DNA contamination are recommended.

2. Gently pipette to homogenize and perform brief microcentrifugation;

3. Incubate at 55 ℃ for 15 min; Note: Pre-incubate at 25 ℃ for 10 min if target RNA lacks Poly(A) tails.

4. Terminate the reaction by incubation at 85 ℃ for 5 min after reverse transcription;

5. Place synthesized cDNA solution on ice for downstream experiments, or store immediately at -20 ℃.

 

Precautions:

This master mix contains pre-mixed Oligo(dT)20VN and random primers, compatible with Poly(A)-tailed eukaryotic mRNA, as well as Poly(A)-free templates including prokaryotic RNA, eukaryotic rRNA and tRNA. It is not applicable for small RNA templates such as miRNA.

 

Product Information

Catalog No.	Product Name	Specifications
abs60246	All-in-One 3rd-Gen First-Strand Synthesis Master Mix	100 Reactions

 

We also recommend First-strand cDNA Synthesis Mix With gDNA Remover (abs601510), a cDNA first-strand synthesis premix with genomic DNA elimination module. Product link: https://www.absin.net/1-cdna/abs601510.html

 

Stage 3: Quantitative Real-Time PCR — The Ultimate Tool for Precise Gene Quantification

 

Quantitative real-time PCR (qPCR) quantifies cDNA templates by real-time monitoring fluorescent signals during amplification, serving as the gold standard for evaluating gene expression levels. Reliable data requires standardized reaction setup, optimized thermal cycling programs and rigorous result analysis.

 

Standard Operation PROTOCOL for SYBR Advanced qPCR SuperMix Kit(abs60087)

 

I. Experimental Guidelines:

1. SYBR Advanced qPCR SuperMix Kit is optimized for two-step qPCR protocols: denaturation at 95 ℃, combined annealing/extension at 60 ℃, compatible with primers with Tm values below 60 ℃;

2. For maximum amplification efficiency in SYBR Green-based real-time PCR, target amplicon length is recommended to range from 60 to 200 bp;

3. A hot-start incubation step at 95 ℃ for 2 min is mandatory to activate hot-start DNA Polymerase before amplification;

4. Recommended total reaction volume is 20 μ L for 96-well block real-time PCR instruments, and 10 μ L for 384-well block instruments.

 

II. Operating Procedures:

1. Thaw and homogenize 2×SYBR Advanced qPCR SuperMix, template gDNA/cDNA, forward/reverse primers, ROX Reference Dye (as required) and RNase-Free Water, then prepare reaction mixtures per the table below. Thanks to hot-start polymerase technology, samples do not require continuous incubation on ice during reaction assembly or instrument setup.

 

Component	Volume for 96-well Block	Volume for 384-well Block	Final Concentration
2×SYBR Advanced qPCR SuperMix	10 μ L	5 μ L	1×
ROX Reference Dye (Add as needed)	2 μ L/0.1 μ L	1 μ L/0.05 μ L	1×
Forward Primer	Variable	Variable	0.7 μ M
Reverse Primer	Variable	Variable	0.7 μ M
DNA Template	Variable	Variable	≤100 ng per reaction
RNase-Free Water	To 20 μ L	To 10 μ L	—

2. Thoroughly homogenize the reaction mixture and transfer appropriate volumes into PCR tubes or reaction plates.

3. Add template gDNA or cDNA (≤100 ng per reaction) into each tube/well containing premix. For two-step RT-qPCR, the volume of undiluted reverse transcription cDNA must not exceed 10% of total qPCR reaction volume.

4. Configure thermal cycling programs on the real-time PCR instrument according to the table below. Fluorescent signal data collection should be performed during the combined annealing / extension step.

Step	Temperature	Duration	Ramp Rate	Cycle Number	Remarks
PCR Hot-Start Activation	95℃	2 min	Max/Fast Mode	—	Heat activation of hot-start DNA polymerase
Denaturation	95℃	5 sec	Max/Fast Mode	35–40 Cycles*3	—
Annealing/Extension	60℃*2	10 sec*1	Max/Fast Mode		Fluorescence data acquisition

 

*Notes:

1. Melting curve analysis is a built-in function of real-time PCR instrument software; follow manufacturer’s instrument instructions for execution.

2. If the instrument cannot complete data collection within the designated short duration, adopt the minimum time required by the equipment for signal capture.

3. This temperature is also applicable to all primers with Tm values lower than 60 ℃.

4. Optimal cycle number depends on initial template DNA load.

5. Place PCR tubes/plates on the real-time PCR instrument and initiate the thermal cycling program.

6. Perform melting curve analysis on amplified PCR products.

This analytical module is embedded in all real-time PCR software; regular melting curve detection is strongly recommended to validate amplification specificity of target amplicons.

 

Technical Specifications:

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

2×SYBR Advanced qPCR SuperMix Volume	High ROX / Low ROX (Volume of ROX Reference Dye to Add)
1.7 mL	340 μL / 17 μL

Instruments requiring High ROX Reference Dye: ABI Prism7000/7300/7700/7900HT/ 7900HT Fast, ABI Step One /ABI StepOne Plus, etc.;

Instruments requiring Low ROX Reference Dye: ABI Prism7500/7500 Fast/ViiA7/ QuantStudio 3/5/6 /7, Stratagene MX4000/MX3005P/MX3000P series, MJ Research Chromo4, Opticon (II), Corbett Rotor Gene 3000, etc.;

ROX-Free Instruments: Thermal Cycler Dice Real Time System, Corbett Rotor-gene6000, Bio-Rad CFX series, Roche LightCycler® 480, Qiagen Rotor-Gene Q, Analytik Jena qTOWER series, etc.

 

Product Information

Catalog No.	Product Name	Specifications
abs60087	SYBR Advanced qPCR SuperMix Kit	100 Reactions

Successful RNA expression research relies on seamless linkage of the three core kits: intact high-quality RNA isolated via TriiZol serves as the foundation, efficient reverse transcription acts as the conversion bridge, and precise quantitative amplification via qPCR forms the core detection module. Mastering this integrated workflow of TriiZol extraction, reverse transcription and qPCR together with all critical precautions enables straightforward resolution of common obstacles in RNA expression profiling. A reliable matched reagent portfolio coupled with standardized operating protocols acts as an invaluable research partner, enabling efficient generation of reproducible data and accelerating the delivery of your scientific findings!

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