혈액 RNA 추출 시 마그네틱 비드 방식에서 주의할 점?

Precautions for Magnetic Bead-Based Blood RNA Extraction

1. Sample Collection and Handling

1.1 Anticoagulant Selection

Preferred: EDTA，inhibits calcium-dependent nucleases.

Acceptable: ACD

Avoid: Heparin (inhibits downstream applications)

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 Sample Volume

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 (예를 들어, guanidinium salts)

Maintain samples on ice during processing

2.3 Carrier RNA

Add carrier molecules (예를 들어, 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

에이_260/280: 1.8–2.1

에이_260/230: >2.0

5.2 Integrity Verification

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

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

5.3 gDNA Contamination Check

Include DNase digestion step if required for downstream assays

5.4 저장

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 (예를 들어, heparinized blood requires specialized kits)

공급자

상하이 링쥔 생명공학 유한회사, 주식회사에 설립되었습니다 2016 생체자성재료 및 핵산추출시약 전문제조업체입니다..

핵산 추출 및 정제 분야에서 풍부한 경험을 보유하고 있습니다., 단백질 정제, 세포 분리, 화학발광, 및 기타 기술 분야.

우리의 제품은 많은 분야에서 널리 사용됩니다., 의료 테스트와 같은, 유전자 검사, 대학 연구, 유전자 육종, 등. 우리는 제품을 제공할 뿐만 아니라 OEM도 수행할 수 있습니다., ODM, 그리고 다른 필요. 관련된 필요사항이 있는 경우, 저희에게 연락하게 자유롭게 느끼십시오 .