Bridges Lab Protocols - User contributions [en]

Mendelian Randomization

2026-04-14T01:21:00Z

Davebrid1: Added link to STROBE-MR

= Mendelian Randomization Standard Operating Procedure (SOP) =

This SOP outlines the standardized pipeline for conducting two-sample Mendelian Randomization (MR) analyses, from instrument selection through to causal inference adjudication.

== Biological Plausibility & Gene-Environment Equivalence (G-EE) ==

As outlined in [https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1005013 ''Gene-environment equivalence: The fundamental principle of Mendelian randomization''] (Davey Smith, Hemani, & Ebrahim, 2026), Mendelian Randomization (MR) is a biological question evaluated with statistics, not a statistical pipeline applied blindly to biological data.

Before defining your genetic instruments, explicitly document the biological plausibility of your study by answering the following three criteria. If the Gene-Environment Equivalence (G-EE) is implausible, the statistical robustness checks in the subsequent sections cannot validate the study.

=== The Target Intervention ===
* '''Question:''' What specific real-world environmental or clinical intervention is this MR simulating?
* '''Action:''' Clearly define the hypothetical "Target Trial." If your exposure is a highly complex or broad behavioral trait (e.g., "television watching" or "loneliness") for which no cohesive, targeted clinical intervention exists, you must heavily scrutinize whether MR is the appropriate tool for your research question.

=== Mechanistic Overlap (The G-EE Assumption) ===
* '''Question:''' Do the proposed genetic variants alter biological pathways in the exact same way as the proposed real-world intervention?
* '''Action:''' Justify the biological function of your intended instruments. For example, if evaluating a pharmaceutical-mimicking target (like statins), your SNPs should specifically lower LDL cholesterol via the targeted pathway (e.g., HMG-CoA reductase inhibition), rather than through nonspecific, highly pleiotropic secondary pathways.

=== Timing and Canalization (Developmental Adaptation) ===
* '''Question:''' How does the lifelong nature of genetic exposure differ from a targeted late-life intervention for this specific trait?
* '''Action:''' Acknowledge the dimension of time. Genetic variants exert influence from conception, meaning human biology may adapt or compensate (canalize) during development. Explicitly state in your preregistration that the resulting causal estimates represent the effect of ''lifelong exposure liability''. Document a warning that these effect estimates cannot be directly translated into predicted outcomes for short-term or late-life clinical trials.

== 1. Genetic Instrument Selection ==
Instruments must be robustly associated with the exposure and independent of one another.

=== 1.1 Stringent Criteria (Primary Analysis) ===
For exposures with well-powered GWAS, apply the following strict inclusion criteria:
* '''P-value Threshold:''' <math>p < 5 \times 10^{-8}</math> (Genome-wide significance)
* '''Linkage Disequilibrium (LD) Clumping:''' <math>r^2 < 0.001</math>
* '''Clumping Distance:''' > 10,000 kb
* '''Reference Panel:''' 1000 Genomes European (or population-matched to the GWAS)

=== 1.2 Loose Criteria (Relaxed Analysis) ===
If the stringent criteria yield fewer than 5 Single Nucleotide Polymorphisms (SNPs), the analysis lacks the degrees of freedom required for standard sensitivity tests. In this case, relax the threshold:
* '''P-value Threshold:''' <math>p < 5 \times 10^{-6}</math>
* '''Requirement:''' If this relaxed threshold is used, '''MR-RAPS''' becomes a mandatory primary reporting model due to the guaranteed introduction of weak instruments and measurement error.

== 2. Estimating Instrument Strength ==
Before conducting MR, validate the strength of the genetic instruments to rule out weak instrument bias.

=== 2.1 Variance Explained (R-squared) ===
Calculate the proportion of variance in the exposure explained by each SNP using the Minor Allele Frequency (MAF) and the effect size (<math>\beta</math>):

<math>R^2 = 2 \times MAF \times (1 - MAF) \times \beta^2</math>

''Note: Review the average MAF. If the average MAF is very low (< 0.05), the instruments rely on rare variants, which may have less stable effect estimates.''

=== 2.2 F-Statistics ===
Calculate the individual F-statistic for each SNP to measure instrument strength, where <math>N</math> is the sample size of the exposure GWAS:

<math>F = \frac{R^2 \times (N - 2)}{1 - R^2}</math>

* '''Individual F-statistic:''' Any single SNP with <math>F < 10</math> should be excluded from the standard IVW analysis.
* '''Total/Mean F-statistic:''' Calculate the average F-statistic across all retained SNPs. If the Mean <math>F < 10</math>, the overall instrument is weak, and MR-RAPS must be prioritized.

== 3. Analytical Models to Test ==
Run the following suite of models to assess causality, heterogeneity, and horizontal pleiotropy.

=== 3.1 The Baseline Model ===
* '''Inverse Variance Weighted (IVW):''' The primary meta-analysis of all SNPs.
** Check Cochran's Q statistic. If <math>p > 0.05</math>, report the '''IVW Fixed Effects (FE)''' model.
** If Cochran's Q <math>p < 0.05</math>, switch to the '''IVW Multiplicative Random Effects (RE)''' model to account for heterogeneity.

=== 3.2 Standard Sensitivity Models (The Big Three) ===
* '''MR-Egger:''' Used to detect directional pleiotropy via the intercept.
* '''Weighted Median:''' Provides a valid estimate if up to 50% of the instrument weight comes from invalid (pleiotropic) SNPs.
* '''Weighted Mode:''' Provides a valid estimate if the largest single cluster of SNPs is valid (ZEro InSIDE assumption).

=== 3.3 Advanced Robustness Models ===
* '''MR-PRESSO:''' Run if Cochran's Q is significant. It detects and removes specific outlier SNPs driving horizontal pleiotropy and provides an outlier-corrected estimate.
* '''MR-RAPS:''' Run if Mean F < 10 or if loose inclusion criteria were used. It handles measurement error from weak instruments.
* '''CAUSE:''' Run as the ultimate robustness check to differentiate true causality from correlated pleiotropy (shared genetic architecture).

=== 3.4 Calculating I-squared GX for MR-Egger ===
To determine if the MR-Egger slope is reliable, you must test the No Measurement Error (NOME) assumption by calculating <math>I^2_{GX}</math>. If <math>I^2_{GX} < 0.90</math>, the MR-Egger slope suffers from dilution bias and should not be trusted.

<syntaxhighlight lang="R">
# R Script for calculating I^2_GX using a harmonised TwoSampleMR dataframe ('dat')

calculate_i2gx <- function(dat) {
beta_x <- dat$beta.exposure
se_x <- dat$se.exposure

# Variance of the exposure estimates
var_beta_x <- var(beta_x)

# Mean of the squared standard errors
mean_se_x2 <- mean(se_x^2)

# Calculate I^2_GX
I2gx <- 1 - (mean_se_x2 / var_beta_x)

# Constrain between 0 and 1
I2gx <- max(0, I2gx)

return(I2gx)
}

i2gx_value <- calculate_i2gx(dat)
print(paste("I^2_GX =", round(i2gx_value, 3)))
</syntaxhighlight>

== 4. Decision Matrix: Which Results to Report ==
Use the following adjudication criteria to synthesize the results from the models above.

{| class="wikitable"
|-
! Scenario !! Data Presentation !! Adjudicated Result to Report
|-
| '''1. Clean Signal'''
| F > 10. Cochran's Q p > 0.05. Egger Intercept p > 0.05. CAUSE Causal Model p < 0.05.
| '''IVW (Fixed Effects)'''. All assumptions are met.
|-
| '''2. Balanced Pleiotropy'''
| F > 10. Cochran's Q p < 0.05. Egger Intercept p > 0.05.
| '''MR-PRESSO (Outlier Corrected)''' or '''IVW (Random Effects)'''. Support with Weighted Median.
|-
| '''3. Weak Instruments'''
| Mean F < 10 OR Relaxed criteria (p < 5e-6) used.
| '''MR-RAPS'''. Baseline IVW is vulnerable to weak instrument bias.
|-
| '''4. Directional Pleiotropy'''
| Egger Intercept p < 0.05.
| '''Weighted Mode''' (if I2GX < 0.90) OR '''MR-Egger Slope''' (if I2GX >= 0.90). IVW is discarded.
|-
| '''5. Correlated Pleiotropy'''
| CAUSE model comparison p > 0.05 (Sharing Model wins).
| '''Null Result (Discard Causality)'''. The traits are correlated due to shared genetics, not a direct causal pathway. Any significant IVW results are false positives.
|-
| '''6. Inconclusive'''
| MR-PRESSO removes >30% of SNPs causing power loss, OR Median and Mode significantly contradict each other.
| '''Unresolved / Inconclusive'''. State that genetic evidence is too pleiotropic to reliably disentangle.
|}

==Reproducible Reporting of Results==

In addition to loading code into shareable repository, for our lab Github, once the manuscript is written complete the [https://www.strobe-mr.org/ STROBE-MR checklist] to ensure robust and clear reporting of key aspects of study design. Include this as supplementary data to the submission even if not strictly required by the journal.

Mendelian Randomization

2026-04-14T01:16:24Z

Davebrid1: /* Mendelian Randomization Standard Operating Procedure (SOP) */ Added notes from the recent PLOS paper about biological plausibility

Mendelian Randomization

2026-04-14T00:24:03Z

Davebrid1: Generated intitial SOP for MR analyses

LDLR Genotyping

2026-04-01T12:40:56Z

Davebrid1: Removed table of contents

Modified from https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075

__NOTOC__

* [[SOP - Ethidium Bromide]]
* [[SOP - Electrophoresis]]

== Reagents Needed ==

=== PCR Primers ===
* '''Ldlr Common Forward''': 5'-TAT GCA TCC CCA GTC TTT GG-3'
* '''Ldlr Wild-type Reverse''': 5'-CTA CCC AAC CAG CCC CTT AC-3'
* '''Ldlr Mutant Reverse''': 5'-ATA GAT TCG CCC TTG TGT CC-3'

=== Master Mix ===
* '''DreamTaq Green PCR Master Mix (2X)''' - Thermo Fisher Scientific, Cat# K1081

=== Additional Reagents ===
* Nuclease-free water
* Template DNA ([[Preparation of Tail Samples (for Genotyping)|tail lysate]] or purified genomic DNA)

== Primer Preparation ==

=== Stock Primer Preparation (100 µM) ===
# Resuspend each lyophilized primer in nuclease-free water to 100 µM concentration. Look on the tube from IDT, for example if there is 23.7 nmoles then you would resuspend in 237 µL of water
# Store at -20°C

=== Primer Master Mix (10 µM each primer) ===
For a 1 mL primer master mix:
* 100 µL of 100 µM Primer 19799
* 100 µL of 100 µM Primer 19800
* 100 µL of 100 µM Primer oIMR7770
* 700 µL nuclease-free water

'''Final concentrations in primer master mix:''' 10 µM each primer

Store at -20°C

== PCR Master Mix Preparation ==

=== Per Reaction (25 µL total volume) ===
* 12.5 µL DreamTaq Green PCR Master Mix (2X)
* 3.75 µL Primer Master Mix (10 µM each)
* 7.75 µL nuclease-free water
* 1 µL template DNA

'''Final primer concentrations in PCR:''' 1.5 µM each primer

=== For Multiple Reactions ===
Prepare master mix for (n+1) reactions, where n = number of samples:
* DreamTaq Green PCR Master Mix (2X): 12.5 µL × (n+1)
* Primer Master Mix: 3.75 µL × (n+1)
* Nuclease-free water: 7.75 µL × (n+1)

Aliquot 24 µL of master mix per tube, then add 1 µL template DNA to each

== PCR Cycling Program ==

This program takes 1:50 to run.

{| class="wikitable"
|+ '''Ldlr''' PCR Program
|-
! Step !! Temperature (°C) !! Time !! Cycles !! Notes
|-
| Initial Denaturation || 94 || 3 min || 1 ||
|-
| '''Touchdown Phase''' || || || ||
|-
| Denature || 94 || 30 sec || rowspan="3" | 10 || rowspan="3" | Decrease annealing temp by 0.5°C per cycle
|-
| Anneal || 65→60 || 30 sec
|-
| Extend || 68 || 45 sec
|-
| '''Standard Cycles''' || || || ||
|-
| Denature || 94 || 30 sec || rowspan="3" | 28 ||
|-
| Anneal || 60 || 30 sec ||
|-
| Extend || 72 || 45 sec ||
|-
| Final Extension || 72 || 5 min || 1 ||
|-
| Hold || 4 || ∞ || ||
|-
|}

== Expected Results ==

* '''Wild type:''' 351 bp
* '''Heterozygote:''' 179 bp AND 351 bp
* '''Mutant:''' 179 bp (GC-rich band)

'''Note:''' Run on 2% agarose gel. The mutant band (179 bp) is GC-rich and may appear fainter than expected.

[[ Category:Genotyping ]]
[[ Category:Mouse Work ]]
[[ Category:PCR ]]

Preparing an Agarose Gel

2026-03-27T12:45:48Z

Davebrid1: Added SOP

[[ SOP - Ethidium Bromide ]]

==Materials==
*Agarose
*TAE Buffer (1X): Dilute from 100X stock
*Thermo Apparatus (Mold, Holder, Comb and Gel Rig)

==Prepare Gel==
#Place plastic mold securely in holder
#Put well comb in mold
#Weight out 2.0g agarose and add 100 mL of TAE (2% solution).
#Microwave 90s until dissolved
#Add 2 uL Ethidium Bromide or Gel Red. Swirl to mix and eliminate sediments
#Pour into mold, add the comb, and wait for it to solidify
#Place gel in cold room if in a hurry
#After loading, run gel at 100V

Preparing an Agarose Gel

2026-03-27T12:44:06Z

Davebrid1: /* Prepare Gel (BioRad Gel Rig) */ Updated instructions

==Materials==
*Agarose
*TAE Buffer (1X): Dilute from 100X stock
*Thermo Apparatus (Mold, Holder, Comb and Gel Rig)

==Prepare Gel==
#Place plastic mold securely in holder
#Put well comb in mold
#Weight out 2.0g agarose and add 100 mL of TAE (2% solution).
#Microwave 90s until dissolved
#Add 2 uL Ethidium Bromide or Gel Red. Swirl to mix and eliminate sediments
#Pour into mold, add the comb, and wait for it to solidify
#Place gel in cold room if in a hurry
#After loading, run gel at 100V

QPCR

2026-03-23T20:08:13Z

Davebrid1: Formatting for GCal link

__NOTOC__
==Real Time qPCR==
===Materials===
*cDNA: see [[First_Strand_cDNA_Synthesis_(AB_Kit)|First Strand cDNA Synthesis (AB Kit)]] for details
*SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])
*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)
*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks- the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example- this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]

===Plate Preparation===
#Book 2h on qPCR machine by signing up on the [https://calendar.google.com/calendar/u/0?cid=Y19jN2RhYmJlMjJiNmQ1YTY0YjAzYzU1NzVmOGUxNmEyZmZjNjAwOTJjYWIyZmYwM2VjY2M1NjkyNzljYmQwY2QzQGdyb3VwLmNhbGVuZGFyLmdvb2dsZS5jb20 Google Calendar]
#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.
#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA's
#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 20-25% more than you need.
#Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.
#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.
#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal. Apply cover by starting in the middle of plate, working out towards edges, and finish by applying high pressure to edges, ensuring a proper seal.
#You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.
#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

===Run Protocol===
* Set up run using [[Set up qPCR Run on Thermo Cloud and QuantStudio|Thermo Cloud/QuantStudio]] or [[Set up qPCR Run on Roche LightCycler|Roche LightCycler]]
Briefly, log into thermo cloud
*Select "Design and Analysis New, qPCR" from the home screen
*Select "set up plate"
*Select "comparative CT-SYBR"
*Correct volume to read, "10.0uL" instead of "20.0uL"
*Select "Plate set up"
*Navigate to the drop down menu marked "passive reference" and select "none."
*Then import [https://docs.google.com/spreadsheets/d/e/2PACX-1vQpOEYWkqKe6tELqIerA7fcwe2LoEQy_8ANGuhOppmGXbD0sBGvKXHnR3z7aYnfEF7--r4FbOd6yCYM/pub?output=csv plate setup] with linked CSV (MUST be a csv file).
*Save to the cloud and upload on the machine when you insert the plate.

==Calculations==
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations

[[ Category: Molecular Biology ]]
[[ Category: RNA ]]
[[ Category: qPCR ]]
[[ Category: Transcription ]]
[[ Category: Expression ]]

QPCR

2026-03-23T20:05:39Z

Davebrid1: Updated calendar link

__NOTOC__
==Real Time qPCR==
===Materials===
*cDNA: see [[First_Strand_cDNA_Synthesis_(AB_Kit)|First Strand cDNA Synthesis (AB Kit)]] for details
*SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])
*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)
*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks- the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example- this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]

===Plate Preparation===
#Book 2h on qPCR machine by signing up on the sheet in room 7013 and on the [https://calendar.google.com/calendar/u/0?cid=Y19jN2RhYmJlMjJiNmQ1YTY0YjAzYzU1NzVmOGUxNmEyZmZjNjAwOTJjYWIyZmYwM2VjY2M1NjkyNzljYmQwY2QzQGdyb3VwLmNhbGVuZGFyLmdvb2dsZS5jb20]
#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.
#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA's
#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 20-25% more than you need.
#Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.
#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.
#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal. Apply cover by starting in the middle of plate, working out towards edges, and finish by applying high pressure to edges, ensuring a proper seal.
#You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.
#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

===Run Protocol===
* Set up run using [[Set up qPCR Run on Thermo Cloud and QuantStudio|Thermo Cloud/QuantStudio]] or [[Set up qPCR Run on Roche LightCycler|Roche LightCycler]]
Briefly, log into thermo cloud
*Select "Design and Analysis New, qPCR" from the home screen
*Select "set up plate"
*Select "comparative CT-SYBR"
*Correct volume to read, "10.0uL" instead of "20.0uL"
*Select "Plate set up"
*Navigate to the drop down menu marked "passive reference" and select "none."
*Then import [https://docs.google.com/spreadsheets/d/e/2PACX-1vQpOEYWkqKe6tELqIerA7fcwe2LoEQy_8ANGuhOppmGXbD0sBGvKXHnR3z7aYnfEF7--r4FbOd6yCYM/pub?output=csv plate setup] with linked CSV (MUST be a csv file).
*Save to the cloud and upload on the machine when you insert the plate.

==Calculations==
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations

[[ Category: Molecular Biology ]]
[[ Category: RNA ]]
[[ Category: qPCR ]]
[[ Category: Transcription ]]
[[ Category: Expression ]]