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Experiments

Interactive guide outlining core experimental workflows — from RNA preparation to imaging analysis in ALS therapeutics research.

RNA Preparation & Reverse Transcription

Isolation of RNA, assessment via Nanodrop, and conversion to cDNA for downstream qPCR analysis.

Transfection & qPCR Analysis

Delivery of antisense oligonucleotides into neuronal cells and quantification of target transcripts.

Aptamer Staining & Imaging

Visualization of RNA aptamer binding and cellular localization using fluorescence microscopy.

RNA Preparation & Reverse Transcription

Nanodrop Analysis of RNA

  1. Blank with dilution buffer.
  2. Load 1 µL sample per spot.
  3. Record concentration and 260/280, 230/280 ratios (>1.9 acceptable).
  4. Use concentrations to calculate RNA volume for downstream reactions.

Reverse Transcription (ProtoScript® II)

Reagents

  • Total RNA (X µg)
  • Oligo(dT)₂₀ primer (X µL)
  • dNTP Mix, 10 mM each (X µL)
  • 10X Reaction Buffer (X µL)
  • 0.1 M DTT (X µL)
  • RNase Inhibitor (X µL)
  • ProtoScript® II Reverse Transcriptase (X µL)
  • Nuclease-free water to X µL

Procedure

  1. Thaw reagents on ice and mix RNA, primer, dNTPs, and water.
  2. Add buffer, DTT, RNase inhibitor, reverse transcriptase, water. Quick spin.
  3. Thermocycle: 25°C 5 min → 42°C 1 hr → 12°C hold.

Transfection & qPCR Analysis

Lipofection with Antisense Oligonucleotides

Reagents

  • ASO (10× stock)
  • OPTI-MEM (100 µL)
  • Lipofectamine 3000 (1 µL)

Procedure

  1. Prepare Master Mix: 100 µL OPTI-MEM + 1 µL Lipofectamine 3000.
  2. Add 7.5 µL ASO per 300 µL Master Mix. Incubate 10 min.
  3. Add 100 µL to each well (final 5 µM).
  4. Incubate; monitor at 24 hr and 5 days.

qPCR Reaction Setup

Reagents per 10 µL

  • 4.5 µL ddH₂O + diluted cDNA (8 ng/µL)
  • 5.0 µL SYBR Master Mix
  • 0.25 µL Forward primer (10 µM)
  • 0.25 µL Reverse primer (10 µM)

Procedure

  1. Dilute cDNA (M1V1 = M2V2).
  2. Mix reagents and plate 10 µL per well.
  3. Run thermocycler according to assay.

Primer Efficiency Curve

  • Dilutions: 8, 4, 2, 1, 0.5, 0.25 ng (1:2 serial)

Procedure

  1. Prepare serial cDNA dilutions.
  2. Plate in triplicates and run qPCR to determine efficiency.

Aptamer Staining & Imaging

Cell Fixation and Aptamer Staining

Fixation

  • Prepare 50 mL: 10 mL 20% PFA + 40 mL ddH₂O.
  • Fix cells 15 min on rocker.

Permeabilization

  • PBST 15 min, wash PBS 3×5 min.

Blocking

  • PBS + 5% donkey serum + 1 µL hsDNA, 45 min on rocker.

Primary Aptamer Binding

  • Blocking buffer + 0.67 µM aptamer overnight at 4°C.

Aptamer Assay – Day 2

  1. Pre-block 5 mL 1% BSA + 3% Triton-X, 30 min RT.
  2. Blocking: NeutrAvidin 10 µg/mL, 15 min.
  3. Wash PBS 3×5 min.
  4. Saturation: D-biotin 1 µL stock + 999 µL PBST, 15 min.
  5. Wash PBS 3×5 min.
  6. Stain: NeutrAvidin–DyLight 550 + 1% BSA, 60 min RT.
  7. Wash PBS 3×5 min.
  8. Counterstain: DAPI 1:1000, 5 min.

Imaging (SH-SY5Y Cells)

  1. Stain cells as above.
  2. Wash PBS 3×5 min.
  3. Counterstain with DAPI 1:1000, 5 min.
  4. Image under fluorescent microscope.