Complete Workflow for RNA Extraction, Purification, and Integrity Assessment

Obtain high-quality RNA suitable for reverse transcription, RT-qPCR, RNA-seq, and hybridization analysis from cells, tissues, and biological samples.

Experiment Objective

To obtain high-quality RNA suitable for reverse transcription, RT-qPCR, RNA-seq, and hybridization analysis from cells, tissues, and biological samples.

Scope and Experimental Requirements

  • Sample: Cultured cells
  • Sample: Fresh or frozen tissue
  • Sample: Blood and body fluids
  • Sample: Plants, microorganisms, and low-abundance samples
  • Difficulty and time consumption: Medium, approximately 1–3 hours
  • Safety: RNase control throughout the process; biosafety management of samples according to origin; strong denaturants and organic solvents used in accordance with chemical regulations.

Standard Workflow

  1. Rapid stabilization or freezing of samples
  2. Thorough homogenization and strong denaturing lysis
  3. Separation and binding of RNA
  4. On-column or solution DNase treatment
  5. Thorough washing and drying of membrane or magnetic beads
  6. Low-volume elution and on-ice operation
  7. Determination of concentration, purity, and integrity
  8. Aliquot and store at low temperature

Equipment, Reagents, and Consumables

  • Equipment: Biosafety cabinet or clean operating area, microcentrifuge, tissue homogenizer, nucleic acid quantification and fragment analysis equipment
  • Reagents: RNA lysis and denaturation solution, reducing agent and RNase inhibitor, binding, wash, and elution buffers, DNase and reaction buffer
  • Consumables: RNase-free columns or magnetic beads, RNase-free centrifuge tubes and filter tips, disposable grinding consumables, low-binding storage tubes

Required Controls and QC

  • Extraction blank
  • RNA positive process control
  • -RT control
  • Exogenous spike-in (as needed)
  • Extraction blank and -RT control negative; concentration and purity meet application requirements; RIN, fragment distribution, or electrophoretic integrity meet preset standards; RT-qPCR internal reference repeatability is acceptable.

Key Parameters and Result Interpretation

Sample ex vivo time, stabilization method, homogenization efficiency, RNase control, DNase treatment, elution volume, and freeze-thaw cycles are key variables.

RNA quality should be judged by purity, integrity, genomic DNA residue, and actual reverse transcription and amplification performance; acceptable thresholds for different samples need to be defined by the method.

Common Problems

When degradation occurs, shorten processing time and strengthen RNase control; when yield is low, improve homogenization or match input amount; when DNA residue is present, optimize DNase; when downstream inhibition occurs, increase washing or secondary purification.

Product BOM Entry

Submit product and technical inquiry for this experiment

Note for Research Use: This workflow is for research experimental design and product selection only; specific parameters, compatibility, specifications, prices, and delivery are subject to project evaluation and formal quotation.