Cad ba chóir aird a thabhairt air sa mhodh coirníní maighnéadach d'eastóscadh RNA fola?

Precautions for Magnetic Bead-Based Blood RNA Extraction

1. Bailiú Samplach agus Láimhseáil

1.1 Anticoagulant Selection

Preferred: EDTA，cuireann sé bac ar núicléis atá ag brath ar chailciam.

Inghlactha: ACD

Avoid: Heparin (cuireann sé bac ar iarratais iartheachtacha)

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 Toirt Samplach

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 (e.g., guanidinium salts)

Maintain samples on ice during processing

2.3 Carrier RNA

Add carrier molecules (e.g., 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

Nigh 1: Remove salts and contaminants

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

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

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 ratio)

RIN (Uimhir Sláine RNA): ≥7 for most applications

5.3 gDNA Contamination Check

Include DNase digestion step if required for downstream assays

5.4 Stóráil

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 (e.g., heparinized blood requires specialized kits)

Soláthraí

Shanghai Lingjun Biteicneolaíocht Co., Teo.bunaíodh i 2016 atá ina mhonaróir gairmiúil d'ábhair bithmhaighnéadacha agus imoibrithe eastóscadh aigéid núicléacha.

Tá taithí shaibhir againn in eastóscadh agus íonú aigéad núicléasach, íonú próitéine, scaradh cille, ceimileacanacht, agus réimsí teicniúla eile.

Úsáidtear ár gcuid táirgí go forleathan i go leor réimsí, mar thástáil leighis, tástáil ghéiniteach, taighde ollscoile, pórú géiniteach, agus mar sin de. Ní chuirimid ar fáil ach táirgí ach freisin is féidir tabhairt faoi OEM, ODM, agus riachtanais eile. Má tá riachtanas gaolmhar agat, bíodh leisce ort teagmháil a dhéanamh linn .