HCT-116 Cell Culture

• Cell seeding distributes a specific number of cells into a flask or plate for accurate experimental results.
• Cell fixing involves washing cells twice with phosphate-buffered saline to remove dead or unattached cells.
• Cells are then fixed with 5% Trichloroacetic acid and submerged in water twice to remove TCA.
• A low pH denatures proteins, which acid-induced precipitation preserves cellular structure.

What HCT-116 Cells Need to be Happy

• Media needs to have DMEM, 10% fetal calf/bovine serum (FCS/FBS), penicillin/streptomycin, and non-essential amino acids.
• Media should have a stable pH, nutrients like glucose and amino acids, growth factors, and antibiotics to prevent contamination.
• Space/surface - requires adequate space to grow and an extracellular matrix to attach and interact.
• Conditions - needs body-like conditions, a stable pH, adequate temperature, CO2, and humidification to prevent drying out.
• Cells are passaged because they use up all nutrients and cover all space in the flask over time.
• Lack of nutrients and space leads to cell death
• SRB assay measures the viability of cells.

SRB Assay Details

• SRB is conjugated, allowing light absorption.
• The extensive pi system absorbs photons between 490-530nm.
• Light absorption depends on: the number of molecules (concentration) in the solution, the path length and the molecule-specific extinction coefficient.
• Untreated and vehicle controls are used to ensure the vehicle does not affect cell viability.
• Untreated control = identifies cell viability in the absence of treatment for comparison.
• Vehicle control = identifies any effect of the vehicle on cell viability, and both controls should yield similar results if the data is valid.
• Adding Tris base to the wells is the final step before measuring absorbance.
• High pH dissociates SRB bound to proteins in the solution.
• If the plate reader indicates samples have overflowed, readings were outside the spectrophotometer range, so samples should be diluted and re-tested.

Technical Replicates Inconsistencies

• Technical replicate values can be inconsistent due to pipetting inaccuracy during cell or Tris treatments, insufficient resuspension of SRB dye, or excess bubbles affecting absorbance readings.
• Technical replicates are important for evaluating the precision of experimental techniques and determining experiment validity.

SRB Assay Observations

• Cells should be observed: before treatment, before SRB application, after washing off SRB, and after dissolving SRB dye for bubbles.
• Technical replicate = testing the same sample multiple times under identical conditions (e.g., same cells treated under the same conditions in CVA experiment).
• Biological replicate = testing different samples across multiple experiments (e.g., cells seeded on different days from different flasks).

Presenting Cell Viability Assay Data

• Graphs should be high resolution with legible titles and captions.
• Graphs should clearly show what they are representing with clear biological replicates.
• Provide details about controls.
• Axis should start at zero.
• Include error bars.

Experiment Questions

• Is the data reliable considering technical replicates and controls?
• What do the results of the experiment indicate?
• How do the results relate to the aim?
• How do the results relate to overall experiments?
• What are the next steps?

RNA Analysis and Isolation

• RNA Isolation = total RNA is extracted from cells, where a cell lysis buffer breaks down cell walls, releasing proteins, enzymes, and nucleic acids.
• Genomic DNA is removed using a chaotropic agent that creates binding conditions for DNA.
• With RNA precipitation, ethanol is added to promote binding to the silica column membrane.
• Impurities and salts are removed by washing.
• Elution is performed in RNase-free water, releasing the RNA from the membrane.
• The nanodrop is then used for RNA quantification

RNA Quantification

• A260/A280 ratio is used to determine the contamination in sugars, salts, or solvents from buffers.
• A260/A280 < 1.8 indicates protein contamination.
• A260/A280 > 2.5 indicates salt contamination.

cDNA Generation

• Oligo dT primers and RNA are denatured by heating to 70 degrees for 5 minutes.
• cDNA synthesis involves dNTP, RNase inhibitors, and RT enzyme, incubated for 1 hour at 42 degrees.

Controls for cDNA Synthesis

• No-RT negative control includes 10X M-MuLV RT Buffer, RNA sample, nuclease-free H2O, dNTPs, and Oligo dT (no enzyme added) to ensure no DNA contamination.
• cDNA no template control involves 10X M-MuLV RT Buffer, M-MuLV RT Enzyme, nuclease-free H2O, dNTPs, and Oligo dT (no RNA added) to avoid DNA/RNA contamination.

cDNA Generation vs. PCR

• cDNA generation uses RNA as a template, reverse transcriptase, dNTPs, and a single oligo dT primer, producing ssDNA (cDNA).
• PCR uses DNA as a template, DNA polymerase, dNTPs, and primer pair, resulting in dsDNA.

Measuring DNA in Real Time

• Using SYBR Green - intercalating dye binds to double-stranded DNA and emits light when illuminated at a specific wavelength
• qPCR no-template control includes RNAse-free water, primers, buffer, Taq Polymerase, and SYBR Green to check qPCR contamination.
• cDNA no-template control includes RNAse-free water, Oligo dT Primer, dNTPs, RNAse inhibitors, and RT enzyme to check cDNA generation contamination.
• Primers are uniquely designed to complement the sequence of the gene of interest.
• Housekeeping genes are expressed constitutively by different cell types.
• Expression is unaffected by cell stress/treatment and serves as an internal control to normalize qPCR data.

qPCR Mechanism & Ct Values

• Ct (Cycle Threshold) is the number of cycles needed for fluorescence to exceed a detection threshold.
• Lower Ct = more starting DNA or higher gene expression.
• Higher Ct = less starting DNA.
• With 4x more DNA, the tube reaches 1,000,000 amplicons 2 cycles earlier.
• With 8x less DNA = Reaches the same amount 3 cycles late.

qPCR Cycle Process

• Denaturation separates DNA strands at ~98°C.
• Annealing & Extension - Primers bind and DNA polymerase extends at ~45-72°C.
• Exponential DNA amplification doubles the amount of DNA each cycle.

Rules for Ct Values

• Technical Replicates = within ±1 cycle of each other
• No-template Controls (NTC) = should have Ct > 30
• Sample Ct values = should be < 35
• No-RT Controls = should have Ct > 10 cycles higher than the sample.

Identifying Outliers & Errors

• Exclude Ct values that deviate from technical replicates.
• Low Ct in NTC = DNA contamination
• Similar Ct in No-RT Control & Sample = RNA contamination
• High Ct values = poor cDNA synthesis

Melt Curve Analysis

• Confirms PCR specificity, ensures only the target gene is amplified, and detects primer-dimers or non-specific products.
• Single clear peak = specific PCR product.
• Multiple peaks = non-specific products.
• NTC with a peak = contamination.

Valid Data Checklist

• Technical replicates = within range
• Controls = pass all checks
• Melt curve = shows a single peak

Data Validity Check

• Check RNA quality (Nanodrop readings), verify cDNA synthesis (reagents & temp), and confirm qPCR conditions (Primers, reagents, cycling conditions).

Protein Expression Analysis Steps

• Cell seeding
• Cell treatment
• Protein extraction
• Protein quantification
• SDS-PAGE
• Transfer
• Western blot

Protein Extraction:

• Store at -80°C for 72 hours before processing, wash cells with PBS and add lysis buffer to break open the cells, where the lysis buffer contains detergents that disrupt the cell membrane, releasing cellular contents.
• Scrape cells to collect material, transfer the lysate into a tube, centrifuge the lysate and collect the supernatant.
• Centrifugation separates soluble components (proteins) from insoluble debris, while removing insoluble material prevents blocking SDS-PAGE pores.
• Keep samples on ice to reduce protein degradation.
• Use protease/phosphatase inhibitors to prevent enzymatic breakdown.

Protein Quantification

• Preparation of diluted protein standards
• Setting up a 96-well plate
• Staining of proteins
• Absorbance is measured using a spectrophotometer.
• Plotted results quantify protein concentration, ensuring sufficient protein and guarantees equal loading of protein.
• SDS-PAGE separates proteins based on molecular weight.

Steps

• Prepare gels (handcast)
• Stacking gel (pH 6.8, larger pores) aligns proteins before separation.
• Resolving gel (pH 8.8, smaller pores) separates proteins by molecular weight.
• Acrylamide concentration determines the separation efficiency.
• Low % gels are better for high MW proteins, and high % gels are better for low MW proteins.
• Mix protein with sample buffer and reducing agent.
• Proteins are coated with SDS, giving them a uniform negative charge
• Reducing agents (e.g., DTT, β-mercaptoethanol) break disulfide bonds, linearizing proteins
• Boil the sample to denature proteins.
• Load the sample into the gel wells.
• Apply electric current to move proteins through the gel, where migration is based on the size of the proteins (smaller proteins travel faster)

Protein Transfer (Blotting) Purpose

• Transfers proteins from the SDS-PAGE gel onto a membrane (e.g., PVDF or nitrocellulose) for further analysis.

Steps

• Transfer proteins from gel to membrane, where larger proteins require longer transfer times.
• Smaller proteins can migrate through the membrane if transfer time is too long.
• Ponceau stain confirms the transfer.
• Blocking prevents non-specific antibody binding.

Western Blotting Steps

• Blocking: Prevents non-specific binding of antibodies.
• Primary antibody incubation: Binds to the target protein.
• Washing: Removes unbound primary antibody.
• Secondary antibody incubation: Binds to the primary antibody.
• This enhances signal via multiple binding sites and increases detection sensitivity compared to directly conjugated primary antibodies.
• Washing removes excess secondary antibody:
• Detection by chemiluminescence using horseradish peroxidase (HRP) and enhanced sensitivity via signal amplification.