Apa yang perlu diberi perhatian dalam kaedah manik magnet pengekstrakan RNA darah?

Precautions for Magnetic Bead-Based Blood RNA Extraction

1. Pengumpulan dan Pengendalian Sampel

1.1 Anticoagulant Selection

Preferred: EDTA，menghalang nuklease yang bergantung kepada kalsium.

Boleh diterima: ACD

Avoid: Heparin (menghalang aplikasi hiliran)

1.2 Processing Timeline

Process samples immediately after collection

If delayed:

Store anticoagulated whole blood at 4°C for ≤24 h

For long-term storage: Freeze PBMCs or lysate at ≤-70°C

1.3 Jumlah Sampel

Strictly follow manufacturer-recommended volumes

1.4 Mixing Technique

Use gentle pipetting/vortexing to prevent RNA degradation

1.5 Erythrocyte Removal

Ensure complete RBC lysis and removal of hemoglobin residues

2. Lysis Phase

2.1 Cell Lysis

Homogenize until no cell aggregates remain

Adhere to specified incubation time/temperature

2.2 RNase Inhibition

Use fresh lysis buffer containing denaturants (cth., guanidinium salts)

Maintain samples on ice during processing

2.3 Carrier RNA

Add carrier molecules (cth., glycogen) for low-yield samples when specified

3. Magnetic Bead Binding and Washing

3.1 Bead-Nucleic Acid Binding

Critical step: Vortex ≥30 sec immediately after adding beads

Incubate for exact duration per protocol

3.2 Magnetic Separation

Place tubes in magnetic stand for ≥2 min until solution clears

Aspirate supernatant without contacting bead pellet

3.3 Washing Procedure

Use freshly prepared ethanol-based wash buffers

Wash 1: Remove salts and contaminants

Wash 2 (optional): Kit-specific buffer for purity enhancement

Final wash: Ensure complete ethanol removal (air-dry beads 2-5 min)

4. Elution Phase

4.1 Elution Conditions

Use nuclease-free water or TE buffer (pH 8.0)

Incubate at 55–65°C for 5 min with periodic mixing

4.2 RNA Recovery

Perform final magnetic separation

Transfer supernatant to new RNase-free tube

5. Quality Control and Storage

5.1 Purity Assessment

A_260/280: 1.8–2.1

A_260/230: >2.0

5.2 Integrity Verification

Electrophoresis: Distinct 28S/18S rRNA bands (2:1 nisbah)

RIN (RNA Integrity Number): ≥7 for most applications

5.3 gDNA Contamination Check

Include DNase digestion step if required for downstream assays

5.4 Penyimpanan

Aliquot RNA

Store at –80°C; avoid >3 freeze-thaw cycles

Critical Reminders

EDTA tubes are essential for nuclease inhibition

Immediate processing prevents RNA degradation

Complete ethanol removal is mandatory before elution

RNase-free environment: Gloves, barrier tips, and decontaminated surfaces

Validate kit compatibility with sample type (cth., heparinized blood requires specialized kits)

Pembekal

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