Lab Safety: Essential Guidelines

Lab Safety Guidelines

• Wear appropriate personal protective equipment, including a lab coat and closed-toe shoes, at all times.
• If necessary, use gloves, safety glasses, and face masks.
• Always remove lab coats and gloves when leaving the work area.
• Remove gloves before answering the phone, opening doors, or using the lift.
• Do not consume or store food/beverages in labs or chemical storage areas.
• Cosmetics should also not be applied.
• Know the hazards of the chemicals being used.
• Consult Material Safety Data Sheets (MSDS) before using unfamiliar chemicals.
• Treat unknown materials as toxic and mixtures as more toxic than their most toxic component.
• Clean up all spillages immediately using appropriate neutralization and disinfection.
• Keep work areas clean and uncluttered.
• Clean up the work area after completing operations or at the end of the day.
• Know the location and use of emergency equipment like First Aid boxes, safety showers, fire extinguishers, and fire alarms.
• Exercise caution when leaving unattended operations.
• Post warning signs, anticipate potential equipment failures, and provide containment for hazardous chemical releases.
• Label all chemical containers with contents, concentration, preparation date, and hazard warnings avoiding abbreviations.
• Report all accidents, even if they seem insignificant, to the demonstrator and technical staff.
• Accident Report Forms are available from the School Office.
• Wash hands frequently to minimize chemical exposure through ingestion and direct contact with the skin, and always before leaving the laboratory.
• Dispose of laboratory waste in the appropriate bins.
• Keep laboratories clean and tidy.
• Passage ways must not be blocked by equipment, boxes, or bags.

Introduction: Differential Gene Expression

• Differential gene expression is central to animal development.
• All cells possess the same genome, but different genes are expressed in different tissues at different developmental stages.
• Understanding animal development involves learning where genes are expressed, how key gene expression is initiated, and how expression changes over time.
• Gene expression has spatial (specific cells, tissues, or structures) and temporal (developmental stage) components.
• One of the first tasks is recognizing when a gene is expressed.
• Several methods are available to study gene expression, including detecting Gene X's products like mRNA or Protein X.
• CELB30010 practicals will focus on detecting gene expression at the mRNA level.
• Methods to detect mRNAs include northern blotting, in situ hybridization, microarrays, RNA-Seq, and RT-PCR (Reverse Transcriptase-PCR).
• RT-PCR is the chosen technique for CELB30010 due to its straightforwardness for early-stage biologists, speed, cost-effectiveness, sensitivity, building on previous PCR experience, and widespread use in biology.
• RT-PCR can test the expression of Your favourite gene (Yfg) if the nucleotide sequence and appropriate RNA are accessible.
• The first step in any RT-PCR experiment is to isolate high-quality RNA, which will be covered in Practical 1.
• Practical 2 focuses on how to assess the quality and quantity of the isolated RNA.
• Practical 3 considers the use of RT-PCR in gene expression studies.
• Practicals 4 and 5 use the RT-PCR approach to examine tissue-specific gene expression.
• Week 6 involves an examination that tests comprehension of the material and interpreting results, comprising 25% of the final CELB30010 grade.

Practical 1: Extraction of Total RNA from cultured cells

• Successfully isolate total RNA from cultured N2a cells
• Understand the importance of minimizing contamination of RNA with Ribonucleases (RNases) during extraction.

Extraction Background Information

• The first step in RNA isolation is cellular disruption, which must be fast and thorough.
• RNA is extremely sensitive to RNAses; therefore, slow disruption may result in RNA degradation by endogenous RNases.
• Incomplete disruption leads to a decreased yield because RNA remains trapped in intact cells.
• Samples are placed in RNA STAT-60TM, a solution containing Guanidine Thiocyanate, a protein denaturant, and are homogenized with a pipette tip (or mechanical homogenizer for tissue samples).
• The RNA STAT-60TM solution also contains phenol to help segregate proteins from RNA, forming an organic phase.
• Chloroform gets added to the mixture after homogenization
• Centrifugation separates the organic and aqueous phases
• Bromochloropropane (BCP) is a less toxic substitute for chloroform.
• RNA remains in the upper aqueous phase and gets easily separated.
• The RNA is then precipitated from the solution by the addition of alcohol.
• The precipitated RNA is collected via centrifugation. After, An appropriate volume of RNase-free H2O or buffer is added to re-suspend the pellet.

RNA Extraction - Special Precautions

• Preventing the introduction of RNases into samples is essential when working with RNA.
• RNA is very susceptible to degradation because it is a single-stranded nucleic acid and, RNases are very active and stable enzymes.
• All plastic ware must be RNase-free and solutions prepared with water treated with DEPC (diethyl pyrocarbonate), an RNAse inhibitor.
• Wearing gloves at all times avoids contamination of samples, with dust particles, bacteria, or RNases.

RNA extraction Protocol Safety considerations

• This protocol uses solutions containing Phenol and other hazardous chemicals, therefore wear gloves

RNA extraction protocol

• Remove ALL tissue culture medium from the cells on the plate
• Add 1.2 ml of RNA STAT-60 TM reagent (in a tube labelled RS) onto the cells. Ensure the entire plate surface is covered by swirling.
• Pass the lysate through a P1000 pipette tip several times.
• Transfer 600 µl of the lysate to a 1.5 ml microfuge tube and incubate at room temperature for 5 min.
• Add 60 µl of BCP (bromochloropropane) to the tube with the cell lysate and close the lid TIGHTLY.
• Shake vigorously for 15 sec.
• Incubate for 10 min at room temperature.
• Place the tubes in the centrifuge and spin for 10 min at 14,000 rpm at 4°C.
• Transfer 250 µl of the upper aqueous phase into a new 1.5ml tube using a P200 pipette (2x125 µl), being careful not to disturb the aqueous/organic interface
• Add an equal volume (250 μl) of Isopropanol.
• Vortex briefly and incubate for 10 min at room temperature
• Microcentrifuge tube spinning at 14,000 rpm for 10 min at 4°C will result in the RNA forming a small pellet at the bottom of the tube
• Remove and discard the supernatant carefully, using a P200 pipette, without dislodging the pellet
• Add 500 µl of 75% Ethanol to wash the RNA pellet.
• Vortex to dislodge the pellet from the bottom of the tube and centrifuge for 5 min at 14,000 rpm at 4°C.
• Remove all the ethanol using a P200 pipette, and allow the pellet to dry at room temperature for 5 min.
• Add 50 µl of RNAse-free water to re-suspend the pellet, and incubate the tube in the heating block at 55°C for 10 min.
• Transfer 5 µl of RNA into a new Eppendorf tube.
• Place both tubes on ice and label clearly with group number and the date
• Samples will be stored at -80°C until the next practical session, where the quantity and quality of the RNA extracted will be assessed.

Practical 2: Assessment of quantity and quality of total RNA

• Determine the quality and quantity of RNA extracted in Practical 1 using Agarose Gel Electrophoresis, spectrophotometry (Nanodrop analysis), and capillary electrophoresis (Bioanalyzer).

Assessment Background Information

• The ability to detect the expression of a gene hinges on isolating its RNA in sufficient quantity and in an undegraded state.
• After isolating RNA from cells/tissues, measuring the quantity, integrity, and purity of the recovered RNA is vital.
• These procedures are critical before using the RNA for potentially time-consuming/expensive downstream applications.

Spectrophotometry

• RNA absorbs UV light at a wavelength of 260 nm, and the amount absorbed is directly proportional to the RNA concentration.
• Measuring the absorbance at 260 nm of the RNA solution can determine the concentration and calculate the amount of RNA recovered.
• An absorbance of 1.0 at this wavelength corresponds to an RNA concentration of 40 µg/ml.
• The relative purity of the RNA sample gets determined through protein concentration, because proteins absorb UV light at 280 nm.
• The ratio between the absorbance at 260 nm and 280 nm is used to measure the presence of contaminating proteins.
• Micro-volume spectrophotometers measure absorbance using very small sample volumes (~1-2 µl).
• These instruments evaluate nucleic acid samples at several wavelengths, providing RNA/DNA concentrations and other quality parameters.

Agarose Gel Electrophoresis

• Agarose gel electrophoresis separates molecules based on charge and size.
• RNA (and DNA) molecules are negatively charged in solution.
• The negatively charged nucleic molecules migrate through the gel to the positive pole when an electric current is applied.
• Smaller fragments migrate faster than larger ones.
• The distance a nucleic acid molecule migrates is a function of its size.
• Molecular weight standards are electrophoresed to ensure the procedure goes well and to allow for size determination.
• The resolving power: ~100 bp to ~40,000 bp (40 Kb).
• Molecules greater than 40 Kb aren't resolved and appear as a diffuse band towards the 'top' of the gel.
• Nucleic acids are invisible and are made visible by dyes that interact with nucleic acids and fluoresce when exposed to UV light.
• Historically, ethidium bromide (EtBr) was used, but less-toxic dyes like SybrSafe are now used.
• Assessing integrity: running on a denaturing agarose gel is the most traditional way.
• Ribosomal RNA (rRNA) is the most abundant form of RNA in the cell.
• RNA extracted from cells shows two sharp, clear bands: 28S rRNA subunit band and, 18S rRNA subunit band
• Intact RNA indicated by a 2:1 ratio (28S:18S)
• Partially degraded RNA has a smeared appearance and lacks sharp rRNA bands
• Completely degraded RNA appears as a very low molecular weight smear.
• Drawbacks includes the Large amount of RNA needed for visualization and, time period involved.
• The practical utilises conventional (non-denaturing) agarose gel electrophoresis, as a faster approach providing information on RNA quality and integrity.

Bioanalyzer analysis: RIN number determination

• The Agilent 2100 Bioanalyzer assesses quantity and quality of RNA samples, utilizing so-called “Lab on a Chip" technology.
• This involves microfluidics, capillary electrophoresis, and fluorescent dyes that bind to nucleic acid.
• A very small amount of RNA (50 ng) is necessary for quality assessment.
• The software evaluates RNA quality by considering the ratio of the two ribosomal bands and the rest of the electrophoretic trace.
• The software produces a RIN (RNA Integrity Number), where 10 indicates a perfect sample and 1.0 indicates the minimum.
• The RIN number is considered the standard objective measure of RNA quality.

Agaraose gel electrophoresis - Protocols

• Obtain Tube from practical 1 containing 5 µl aliquot of the RNA extract
• Add 2 µl of loading buffer (see below) to the sample and mix by pipetting up and down 3 or 4 times.
• Pipette 7 µl of the sample into agarose gel well Care must, that the sample does not contaminate adjacent wells. Record the sample/lane number.
• Place 10µl of the molecular weight standard into an adjacent well.
• Place the cover on the gel apparatus. and switch on the power supply to 130 V
• Monitor the progress by visualizing, by tracking the location of the loading dye.
• Once the orange dye reaches the bottom of the gel (approximately 40 min), switch off the instrument.
• A photograph from the Syngene image analysis system will be provided. Incorporate it into the lab manual.

Agarose gel electrophoresis - LAoding Buffer

• Prior to loading RNA or DNA, samples gets mixed with a loading buffer that contains key reagents:
• Glycerol, increases the density of the sample so that it sinks into well.
• Dye, Orange G, Migrates at the same rate as very small nucleic acids (approx 50 bp), is used as visual marker.
• The blue dye in the molecular weight standard is called Bromophenol Blue.

Agaraose gel electrophoresis - Examination Questions

Before confident that the gel preparation/ electrophoresis performed correctly here are some questions:

• Can you see the all the molecular weight standards?
• Have they resolved into discrete bands, separated from each other?
• Can you identify the molecular weight of each band?

RNA Sample Examination

• The RNA Sample is the sample that has undergone special treatment/testing
• In the lab, it is the sample on the agarose gel:
• Can you see 2 bands corresponding to the Ribosomal RNA subunits?
• Is the top band more intense than the lower band? Are the two bands in a 2:1 ratio?
• Is there any evidence of RNA degradation?
• Is there any evidence of contamination of your RNA with genomic DNA (gDNA)? Where on the gel would you expect to find gDNA? Explain your answer?
• Any other observations?

Spectrophotometry - Protocol

• Measure the RNA solution absorbance from Practical 1 with Nanodrop 1000 instrument.
• Absorbance is referred to as its Optical Density (or OD) at that wavelength.
• Record the values of the OD260, OD 280, OD 260/280 ratio and the RNA concentration (in ng/µl) provided by the spectrophotometer in Table 1.
• Calculate the yield of the RNA preparation from Practical 1 (Total amount of RNA recovered, in µg)

Bioanalyzer analysis: RIN number determination - Protocol

• The electrophoresed and processed RNA will be prepared for you. It may need dilution. if it does, obtain the dilution factor from the demonstrator.
• A copy of the electropherogram and associated data for your RNA sample will be given to you
• Incorporate the data into your CELB30010 lab manual.
• Record the RIN number, the rRNA ratio (28S/18S), the RNA concentration and RNA dilution factor (if relevant) in Table 3.
• Calculate the yield of your RNA preparation from Practical 1 (Total amount of RNA recovered, in µg).

Reflection on outcome of analysis of RNA quantity and quality Questions

• Two methods have been used to measure the yield of RNA, do they give the same answer? Are the values different?
• There are two measured parameters relating to the quality of the isolated RNA. they are, contamination of RNA with protein, and integrity of RNA. Considering this, is the quality of RNA satisfactory?
• As a professional biologist employed in industry or academia, are you prepared to spend a month of my (valuable) time examining gene expression using this RNA? or, Are prepared use this RNA in an experiment that will cost several thousand € (a micro-array examination of gene expression or RNA sequencing experiment, perhaps)?
• Has Examining extracted RNA by a 'low-tech' approach (conventional agarose gel electrophoresis) yieleded any useful information? Does it offer any advantages in the assessment of RNA?

Practical 3: Detecting gene expression by RT-PCR

• Taq polymerase used in PCR requires DNA as a template
• The product of gene expression is RNA. Inorder to examine the gene expression, RNA must be converted into a DNA format
• Reverse transcriptase (RT) is an enzyme that converts to DNA
• Reverse transcriptase is found in viruses that have RNA genomes, and is also known as RNA-directed DNA polymerase.
• RT was discovered by Howard Temin and David Baltimore. They shared the 1975 Nobel Prize in Physiology or Medicine for their discovery.
• Molecular biologists realized that reverse transcriptase can convert mRNA into cDNA, and Combining the Reverse transcriptase into cDNA with the amplification power of PCR makes it quite straightforward to detect expression of any gene
• The term used is RT-PCR.
• This is more convenient than using mRNA, because single-stranded nucleic acid is not as stable as double stranded DNA, and is quite ‘delicate' and easily degraded by RNases.

Mouse Alb gene sequence analysis

• For any gene, a critical step in detecting its expression by RT-PCR is the design of appropriate oligonucleotide primers for the PCR step. This requires careful examination of the mRNA sequence.
• To amplify a portion of the mouse albumin (Alb) transcript from mouse liver cDNA, the Alb gene codes for albumin, the most abundant plasma protein in mammals, and Albumin is expressed in the liver.
• Albumin protein is exported into the blood stream, where it responsible for about 80% of the total osmotic regulation in blood and, it transports fatty acids from adipose tissue to muscle
• cDNA made from liver-derived RNA should therefore provide a convenient target for PCR amplification of Alb.

mRNA Sequence information (Accession numbers)

• Characterized mRNA sequence and nucleotide sequence and other relevant information get submitted to public database
• A submission goes to the National Centre for Biotechnology Information (NCBI) nucleotide database. A unique identification number called an Accession number is given
• In order to get access to any entry, the accession number must be known
• NM_009654 is mouse Alb mRNA and provided for in Figure 4.
• The nucleotide sequence is written in 5' to 3' direction (reading left to right) as with all nucleic acids

Alb mRNA - Accession

• Find the Alb entry in the NCBI nucleotide database:
• Go to http://www.ncbi.nlm.nih.gov/guide/
• Search the nucleotide database using the mouse Alb mRNA accession number. This gets you the entry associated with this number.
• Find the coding sequence (usually abbreviated CDS) and highlight the start/stop codon.
• The sequence codes for the amino acids of the albumin is the open reading frame (ORF).
• Untranslated regions (UTRs) are, 5'UTR and 3'UTR.
• Regions gets referred to as untranslated regions (UTRs), 5'UTR and 3'UTR. Why do you think these sequences are present in the mRNA? Do they have any purpose?

mRNA and Alb Gene sequence comparison

• Genomic DNA of the mouse has been sequenced and is available in a public database.
• Accessing the mouse genome is through the University of California Santa Cruz Genome Bioinformatics site (http://genome.ucsc.edu/).

Comparing the mRNA and Alb Gene sequences

• Open the Blat page of the UCSC site and select the mouse genome from the drop-down list of genome choices.
• Add mRNA sequence and hit Submit, to compare the mRNA sequence with the genomic DNA sequence.
• the Blat search results give two views of your query.
• A visual representation of the location of the Alb sequence along the browser ('browser')
• A detailed view of the comparison of the Alb mRNA with the Alb genomic DNA sequence ('details')
• Chose the latter view and Identify the mRNA sequence (in blue), why do you think there are stretches of black nucleotides between the blue stretches of sequence?
• Use, for the blue sequences and the intervening sequences.
• List, How many exons comprise the Alb gene? How many introns?
• Mark, the Alb exon/exon junctions on the Alb mRNA sequence.

Albumin oligonucleotide primers

• The primer for forward and reverse have been designed to amplify a fragment of the Alb cDNA, and given on the location mRNA sequence.
• An Alb gene sequence can be found in Figure 5, of which exons are in capital letters while the intron sequences are in lower-case font.
• Find the Visible Alb exons in figure and Identify and mark, the location of the oligonucleotide primers

Alb oligonucleotide fragments - Prediction

• From Figure 4 and Figure 5, determine the size of the Alb fragments that you predict will be amplified from a cDNA template or from a genomic DNA template, and note for comparison and analyis.
• Oligonucleotide primers become the ends of the amplified fragment during PCR,

Practical 4, Objective 1: Detecting Alb gene expression by RT-PCR

• Mouse liver cDNA and mouse genomic DNA template used with oligonucleotide primers detailed earlier.
• The PCR products separate on an agarose gel.

Practical 4: Table

• The table contains the sizes of fragments in the molecular weight standard used in the Alb gene expression experiment.
• Check for the molecular weight standards and answer the gel examination questions.

Practical 4: Positive and negative control examination

• Make any conclusions about Alb gene expression once PCR has been done correctly
• Check positive and negative control (target, non-target), as well as any band (size, and if its a size discrepancy, think of possible explanations)

Practical 5, Objectives

• Analyze the products of the PCR experiment set up in Practical 4.
• Confirm that Gapdh provides a reliable indication of RNA/cDNA quality.
• Determine whether the experimental data support the classification of mouse Prp and/or NefH as tissue-specific genes.
• Confirm whether N2a cells express Prp or NefH.

Practical 5 - Agarose gel electrophoresis protocol

• Obtain the PCR products from your demonstrator.
• Pipette 10 µl of each product into an individual well of the agarose gel*., Make sure not to contaminate adjacent wells. Record the sample/lane number. Place 10µl of the molecular weight standard** into an adjacent well
• Place cover on the gel apparatus and Switch on the power supply, setting it at a constant voltage of 130 V
• Monitor the progress of the gel can be monitored visually, by tracking the location of the loading dye(s). When the yellow dye is close to the bottom of the gel switch off,
• Your demonstrator will remove the gel from the tank, photograph it, and incorporate the photograph into your lab manual and examine it as described below.
• Notes, a green PCR buffer which contains, Gylcerol and a blue/ yellow dye. Use Table 5, for The size of the fragments that gets reported