Lab Notebook

July 17: Preparation for qPCR Plate

Sample Assessment

When we observed the samples, we recorded that samples 1, 3, 4 (maybe), 5, 9, 11, 22 (maybe) may be uneven in sample size but we are proceeding with the qPCR.

Volume Calculations

Completed all calculations for plating. Using the amount of cDNA (245 µM) calculated previously, we were able to determine how much µL of ddH₂O each of the replicates required for both the +dox and -dox samples.

Reaction Composition

We are going to pipette 10 µL in each reaction:

• 4.5 µL is going to be ddH₂O + cDNA
• 5.0 µL is Poner Syber MM
• 0.5 µL is forward and reverse primers at 10 µM
  • 0.25 µL Forward + 0.25 µL Reverse

cDNA Dilution Calculation

Using M₁V₁ = M₂V₂ we were able to determine that 0.14694 µL is required to dilute the 245 ng of cDNA into 4.5 µL of 8.0 ng/µL cDNA.

Upcoming plans

Now that we finished labeling and setting up our qPCR plate, we are going to pipette samples and run the experiment

July 18: Running qPCR Experiment

Sample Volume Verification

First we needed to redo our calculations and also account for the discrepancies in the samples of cDNA that had less than ideal amounts. Using a pipette, we measured how many µL of cDNA were actually in the following samples:

Sample 1: 11 µL

Sample 3: 11 µL

Sample 4: 12 µL

Sample 5: 15 µL

Sample 9: 13 µL

Sample 11: 8 µL

Sample 22: 10 µL

We are assuming that all the other samples have around 20 µL of cDNA.

Calculation Example (Sample 14)

Based on our qPCR setup with METTL3, YTHDF2, and a control group, we calculated how much cDNA and DDH₂O each reaction contained.

For example, the first group, 14, testing METTL3, cryptic exon, full exon, and GAPDH:

• Has 3 technical replicates → total of 15 reactions
• Added 3 extra reactions for pipetting error → 18 reactions total
• 8 ng cDNA per reaction × 18 reactions = 144 ng total cDNA needed
• 5.0 µL Poner Syber MM × 18 = 90 µL
• 0.5 µL primer × 18 = 9 µL

Calculating cDNA volume needed:

144 ng ÷ 245 ng/µL × 20 µL = 11.75 µL of cDNA stock

Total reaction volume calculation:

10 µL × 18 reactions = 180 µL total

180 µL - 90 µL (MM) - 9 µL (primer) - 11.75 µL (cDNA) = 69.25 µL of DDH₂O

We repeated the above for all the wells in the chart and accounted for the lower amounts of cDNA in the listed numbers above.

Next Steps

Next visit, finish adding the ddH₂O based on the calculations on the spreadsheet.

July 21: Extract RNA and Complete qPCR

What we did: Extract RNA and complete qPCR

July 22: Nanodrop Analysis

How to Use a Nanodrop

1. Add blanks on all 8 spots (the blank is the same solution the RNA was diluted in)
2. Clean all spots with ddH₂O on a kim wipe
3. Add 1 microliter of each sample to each of the 8 wells, close the nanodrop, and let the system run
4. Ratios 260/280 and 230/280 indicate the purity of the sample; anything above 1.90 indicates low contamination
5. Repeat for all samples

Pipetting Amounts for ProtoScript® II Kit

Determine pipetting amounts:

1. Record all the concentrations determined from the nanodrop
2. Determine total well amount that will result in >1 microliter pipetted for each sample (in this case, it was 265 microliters)

Pipetting amounts per sample:

• RNA: 265/concentration (should be >1 microliter, varies per sample)
• ddH₂O: 6 - RNA volume
• Master mix: We accounted for 2 extra wells for pipetting error
  • 26×2 microliters of Oligo(dT)₂₀ primer
  • 26×2 µL dNTP Mix (10 mM each)
  • 26×10 microliters of reaction buffer

Adding Primer to qPCR Plate

• There are 13 reactions being tested for each protein in rows 1-5, so we multiplied 14 (to account for pipetting error) by 5.5 µL of —- to calculate a total of 77 µL for each: METTL3, TDP43, full exon, cryptic exon, and GAPDH
• We repeated this process for rows 6-10, only replacing METTL3 for YTHDF2, and pipetted another 5.5 µL of — in each corresponding well
• For rows 11-22, the control group, we are testing 23 reactions for each protein (METTL3, YTHDF2, TDP43, full exon, cryptic exon, and GAPDH), so we multiplied 24 reactions (account for pipetting error) by 5.5 µL of — to calculate a total of 132 µL for each proteins — of which we added 5.5 into each corresponding well
• Using a multichannel pipette, we added 5.5 of the respected protein’s—- to each corresponding well

We then ran the qPCR and will analyze the results

July 23: Prepare for qPCR Plate

Updated Calculations

Completed all calculations for plating. Using the amount of cDNA (265 µM) calculated previously, we determined dilution requirements.

Reaction composition (10 µL total):

• 4.5 µL ddH₂O + cDNA
• 5.0 µL Poner Syber MM
• 0.5 µL primers (0.25 µL Forward + 0.25 µL Reverse) at 10 µM

Updated cDNA dilution:

Using M₁V₁ = M₂V₂: 0.135849 µL is required to dilute the 265 ng of cDNA into 4.5 µL of 8.0 ng/µL cDNA.

Calculation Example (Sample 1)

Sample 1, testing METTL3, cryptic exon, full exon, and GAPDH:

• 3 technical replicates = 15 reactions
• Added 3 extra for error = 18 reactions
• 8 ng cDNA × 18 = 144 ng total
• 144 ng ÷ 265 ng/µL × 20 µL = 10.87 µL cDNA needed

Total volume calculation:

180 µL (10 × 18) - 90 µL (MM) - 9 µL (primer) - 10.87 µL (cDNA) = 70.13 µL DDH₂O

July 24: Thermocycling and Plating

Sample Preparation

Samples 12 and 13 did not have enough master mix so we had to re-mix both samples.

Thermocycle program:

• 25°C for 5 minutes
• 42°C for 1 hour
• 12°C on hold

Detailed Calculation Example (SND-1)

Before beginning plating for the qPCR, we calculated cDNA and ddH₂O amounts for each well.

For SND-1 triplicates (6 reactions + 1 extra = 7 reactions):

1. Concentration calculation: 7 reactions × 8 ng/µL = 56 ng/µL total
2. Stock volume needed: 56 ng/µL ÷ 265 µL total cDNA = 0.21132075471
3. From 20 µL stock: 0.21132075471 × 20 µL = 4.23 µL cDNA

ddH₂O calculation for 7 reactions:

• Total volume: 10 µL × 7 = 70 µL
• Components:
  • cDNA: 4.23 µL
  • Poner Syber MM: 5.0 µL × 7 = 35 µL
  • Primers: 0.5 µL × 7 = 3.5 µL
• ddH₂O: 70 - 35 - 3.5 - 4.23 = 27.27 µL

After completing calculations for all reactions 1-24, we mixed the calculated cDNA with ddH₂O to create diluted cDNA mixes, then pipetted 4.5 µL of diluted mix into each corresponding well.

July 29: Master Mix Preparation

Cyber and Primer Calculations

We finished adding 4.5 µL of diluted cDNA to the remaining wells and moved on to adding the cyber and primer master mix.

Primer counts and calculations:

• SND: 57 wells × 1.1 (10% error) = 63 wells
  • Cyber: 5 µL × 63 = 315 µL
  • Primer: 0.5 µL × 63 = 31.5 µL
• FAM: Same as SND (315 µL cyber, 31.5 µL primer)
• DAZ: Same as SND (315 µL cyber, 31.5 µL primer)
• GAPDH: 91 wells × 1.1 = 100 wells
  • Cyber: 5 µL × 100 = 500 µL
  • Primer: 0.5 µL × 100 = 50 µL

Primer Efficiency Curve Setup

We decided to simultaneously run our experiment for establishing a primer efficiency curve to see how well our primers were absorbed to amplify the RNA sequence.

Serial dilutions performed:

Dilution preparation (using samples 14 and 15):

• Mixed samples 14 and 15: total 16 ng
• 4.5 µL per well × 3 wells = 13.5 µL per primer
• 4 primer sets × 13.5 µL = 54 µL (account for 60 µL)
• Serial dilutions require 60 µL × 2 = 120 µL total

Scaling calculations:

• Scaling factor: 120 ÷ 31.5 = 3.80
• DNA: 4.23 µL × 3.87 = 16.11 µL
• ddH₂O: 27.27 µL × 3.87 = 103.89 µL

July 30: qPCR Run and Results

Execution

We ran the qPCR plate, but the experiment failed.

Issues Encountered

• Got many N/A values for primer efficiency curve readings
• Standard deviations were very high (later redone)
• Possible ddH₂O contamination identified

Materials Note

Used Bio-Rad Microseal B Adhesive Sealer