sample preparation in molecular testing.pptx

persiapan sampel untuk pemeriksaan molekuler

1. Persiapan sampel untuk pemeriksaan molekuler

2. Molecular testing A laboratory method that uses a sample of tissue, blood, or other body fluid to check for certain genes, proteins, or other molecules that may be a sign of a disease or condition, such as cancer. Can also be used to check for certain changes in a gene or chromosome that may increase a person’s risk of developing cancer or other diseases. May be done with other procedures, such as biopsies, to help diagnose some types of cancer. May be used to help plan treatment, find out how well treatment is working, make a prognosis, or predict whether cancer will come back or spread to other parts of the body. Also called biomarker testing and molecular profiling. Definition of molecular testing - NCI Dictionary of Cancer Terms - NCI

3. Nucleic acids

4. Gene mutation Both the type of mutation and its location in relation to the gene determine the ultimate effect on the protein product. Mutations may have no effect on protein expression or functional activity; many human proteins exist in detectable variants that have no disease association. Mutations occurring in intron regions presumably have no effect at all. Even small mutations in regions that code for key functional domains (exons) may drastically alter or eliminate function.

5. Gene mutation Nonsense mutations that change an amino acid codon to a stop codon, or vice versa, result in abnormally short or long protein products, respectively. Missense mutations that change the code from one amino acid to another, may or may not alter protein function depending on the change and function of the amino acid. Mutations within sequences that denote intron-exon splice sites could eliminate exons or introduce introns into the transcribed mRNA. Mutations may also occur in regulatory sequences, such as promoters or enhancers, producing dramatic effects on the levels of transcription.

6. Types of mutations

7. Application of molecular diagnosis in cancer Assistance in disease diagnosis and classification Determination of prognosis Determination of therapeutic options, e.g. targeted therapy Combination of the above: In most cases, detection of molecular markers may provide both diagnostic and prognostic information  Techniques commonly used in cancer molecular pathology include detection or identification of specific sequences of genes (deoxyribonucleic acid [DNA] and/ or its transcribed ribonucleic acid [RNA]) or gene product (protein) alterations.

8. Methods Amplification-based techniques (i.e., polymerase chain reaction [PCR], reverse-transcriptase PCR [RT-PCR], branched DNA testing) Hybridization methods (i.e., in situ hybridization, or fluorescence in situ hybridization [FISH]), microarray-based approaches (i.e., comparative genomic hybridization [CGH], DNA, RNA, microRNA [miRNA] microarray) Sequencing. PCR and FISH are among the most commonly used methods, whereas high-throughput techniques, such as array-based tests and next-generation sequencing (NGS) have a promising future but have not been fully established in clinical settings due to the lack of standards.

9. Sample preparation Any operations performed on a sample prior to instrumental analysis, typically consisting of the separation of target analytes from some matrices, the concentration of analytes, and the chemical or physical modifications made to improve downstream separation or detection The typical examples of sample preparation include processes such as dissolving samples in a solvent, extracting analytes from a matrix, separating interfering components of a sample from the target analytes, enriching target analytes to make their detected signal stronger, and reacting analytes with some reagent to convert them into measurable derivatives, while the typical examples of sample pretreatment include changes in physical state such as freezing or crystallizing or grinding of a sample.

10. Sample preparation Adequately developed sample preparation is essential for obtaining a clean sample for analysis and ensuring that the subsequent steps and instrumentation used in an analytical process are not negatively impacted Sample preparation/pretreatment steps of a given method greatly impact the costs, time, and overall success of an analytical process

11. Sample preparation Estimated to account for approximately 66–80% of sample analysis time introduce much of the error in interlaboratory analyses, hinder the identification of sources of error arising from multiple difficulties introduce environmental hazards due to the large volume of hazardous solvents and waste generated present health hazards to technicians or operators involved in a process due to exposure to large volumes of harmful solvents and residues involved in processes such as extraction

12. Nucleic acid extraction principles The genomic DNA (gDNA) and total RNA are frequently the genetic elements targeted for molecular biology experiments since these are the main sources of genetic information in an organism. Type of organisms should be considered: yeast, plants, and bacteria species cells have a cell wall structure  recommended to include an extra step for cell lysis before disrupting the plasma membrane. This step is usually an enzymatic or mechanical process to disrupt the cell wall.

13. Nucleic acid extraction principles Genomic DNA and total RNA extraction requires cell lysis to accomplish the task. The purification of DNA and RNA molecules is required to avoid contaminations with other intracellular components, such as proteins and metabolites. Commonly used methods for DNA and RNA purification: precipitation with phenol-chloroform or isopropanol, or by spin columns with silica membrane.

14. DNA EXTRACTION: 5 basic steps disruption of the cellular structure to create a lysate separation of the soluble DNA from cell debris and other insoluble material binding the DNA of interest to a purification matrix washing proteins and other contaminants away from the matrix and elution of the DNA.

15. Creation of lysate The first step in any nucleic acid purification reaction is releasing the DNA/RNA into solution. The goal of lysis is to rapidly and completely disrupt cells in a sample to release nucleic acid into the lysate. 4 general techniques for lysing materials: physical methods enzymatic methods chemical methods combinations of the three.

16. Creation of lysate: Physical Method Typically involve some type of sample grinding or crushing to disrupt the cell walls or tough tissue. A common method of physical disruption is freezing and grinding samples with a mortar and pestle under liquid nitrogen to provide a powdered material that is then exposed to chemical or enzymatic lysis conditions. Grinders can be simple manual devices or automated, capable of disruption of multiple 96-well plates. Physical methods are often used with more structured input materials, such as tissues or plants. Other devices use bead beating or shaking in the presence of metallic or ceramic beads to disrupt cells or tissues, or sonication to disrupt tissues and lyse cells.

17. Creation of lysate: Chemical method Chemical methods can be used alone with easy-to-lyse materials, such as tissue culture cells or in combination with other methods. Cellular disruption is accomplished with a variety of agents that disrupt cell membranes and denatures proteins. Chemicals commonly used include detergents (e.g., SDS) and chaotropes (e.g., guanidine salts and alkaline solutions) In many protocols, a combination of chemical disruption and another is often used since chemical disruption of cells rapidly inactivates proteins, including nucleases.

18. Creation of lysate: ENZYMATIC METHODS Enzymatic methods are often used with more structured starting materials in combination with other methods with tissues, plant materials, bacteria and yeast. The enzymes utilized help to disrupt tissues and tough cell walls. Depending on the starting material, typical enzymatic treatments can include: lysozyme, zymolase and liticase , proteinase K, collagenase and lipase, among others. Enzymatic treatments can be amenable to high throughput processing, but may have a higher per sample cost compared to other disruption methods.

19. Clearing of lysate Depending on the starting material, cellular lysates may need to have cellular debris removed prior to nucleic acid purification to reduce the carryover of unwanted materials (proteins, lipids and saccharides from cellular structures) into the purification reaction, which can clog membranes or interfere with downstream applications. Usually clearing is accomplished by centrifugation, filtration or bead-based methods. Centrifugation require more hands-on time, but is able to address large amounts of debris. Filtering can be a rapid method, but samples with a large amount of debris can clog the filter. Bead-based clearing, like the method used with Promega particle-based plasmid prep kits, can be used in automated protocols, but can be overwhelmed with biomass. Once a cleared lysate is generated, the DNA can then be purified by many different chemistries, such as silica, ion exchange, cellulose or precipitation-based methods.

20. Binding to purification matrix DNA of interest can be isolated using a variety of different methods: purification by binding to matrices (silica, cellulose and ion exchange) Each of these chemistries can influence the efficiency and purity of the isolation, and each have a characteristic binding capacity. Bind capacity is an indication of how much nucleic acid an isolation chemistry can bind before it reaches the capacity of the system and no longer isolates more of that nucleic acid. We can manipulate the binding conditions to enrich for different categories of nucleic acid (e.g., chemistries that selectively bind RNA versus DNA or large versus small fragments).

21. Purification: Solution-based chemistry Rely on alcohol precipitation. Following the creation of lysate, the cell debris and proteins are precipitated using a high-concentration salt solution. The high concentration of salt causes the proteins to fall out of solution, and then centrifugation separates the soluble nucleic acid from the cell debris and precipitated protein The DNA is then precipitated by adding isopropanol to the high-concentration salt solution. This forces the large genomic DNA molecules out of solution, while the smaller RNA fragments remain soluble. The insoluble DNA is then pelleted and separated from salt, isopropanol and RNA fragments via centrifugation. Additional washing of the pellet with ethanol removes the remaining salt and enhances evaporation. Lastly, the DNA pellet is resuspended in an aqueous buffer like Tris-EDTA or nuclease-free water and, once dissolved, is ready for use in downstream applications.

22. Purification: SILICA-binding chemistry Based on binding of the DNA to silica under high-salt conditions. The key to isolating any nucleic acid with silica is the presence of a chaotropic salt like guanidine hydrochloride. Chaotropic salts present in high quantities are able to disrupt cells, deactivate nucleases and allow nucleic acid to bind to silica. Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column. Once the washes are finished, the genomic DNA is eluted under low-salt conditions using either nuclease-free water or TE buffer. Binding to silica is not DNA specific, so if pure DNA is required, there is also the option to add ribonuclease (RNase A) to the elution buffer. RNA may be may be copurified with gDNA, and the addition of RNase to the elution buffer ensures the removal of the vast majority of contaminating RNA. This chemistry can be adapted to either paramagnetic particles (PMPs), or silica membrane column-based formats. While both methods generally represent a good balance of yield and purity, the silica membrane column format is more convenient Strong magnet for particle capture needed to process the DNA samples

23. Purification Cellulose-Binding Chemistry Nucleic acid binds to cellulose in the presence of high salt and alcohols. The binding capacity of cellulose-based methods is very high. Conditions can be adjusted to preferentially bind different species and sizes of nucleic acid. As a result of the combination of binding capacity and relatively small elution volume, we can get high concentration eluates for nucleic acids. Ion Exchange Chemistry Ion exchange chemistry is based on the interaction that occurs between positively-charged particles and the negatively-charged phosphates that are present in DNA. The DNA binds under low salt conditions, and contaminating proteins and RNA can then be washed away with higher salt solutions. The DNA is eluted under high salt conditions, and then recovered by ethanol precipitation.

24. WASh & Elution 4. Washing Wash buffers generally contain alcohols and can be used to remove proteins, salts and other contaminants from the sample or the upstream binding buffers. Alcohols additionally help associate nucleic acid with the matrix. 5. Elution DNA is soluble in low-ionic-strength solution such as TE buffer or nuclease-free water. When such an aqueous buffer is applied to a silica membrane, the DNA is released from the silica, and the eluate is collected. The purified, high-quality DNA is then ready to use in a wide variety of demanding downstream applications, such as multiplex PCR, coupled in vitro transcription/translation systems, transfection and sequencing reactions.

25. DNA from FFPE Procedure 6 steps :  Remove paraffin: paraffin is dissolved in xylene and removed  Lyse: sample is lysed under denaturing conditions with proteinase K  Heat: incubation at 90°C reverses formalin crosslinking  Bind: DNA binds to the membrane and contaminants flow through  Wash: residual contaminants are washed away  Elute: pure, concentrated DNA is eluted from the membrane

26. RNA Extraction: Sample collection & protection Finding the most appropriate method of cell or tissue disruption for your specific starting material is important for maximizing the yield and quality of your RNA preparation. During sample disruption for RNA isolation, it is crucial that the lytic agent or denaturant be in contact with the cellular contents at the moment that the cells are disrupted  can be problematic when: tissues or cells are hard (e.g., bone) contain capsules or walls (e.g., yeast, gram-positive bacteria, spores) workflows prevent processing immediately after collection (e.g., transport from a site of collection to another location for processing) samples are numerous (making rapid processing difficult). A common solution to these problems is to freeze the tissue/cells in liquid nitrogen or on dry ice. The frozen samples are often preprocessed to select a desired mass or to partially pulverize the sample before exposure to denaturant  complex, time-consuming, and laborious process.

27. RNA Extraction: Sample collection & protection RNA later  and    RNA later -ICE  RNA stabilization solutions allow the researcher to postpone RNA purification for days, weeks, or even months after tissue collection, without sacrificing the integrity of the RNA. Dissected tissue, body fluids, or collected cells are simply introduced into the RNA later  solution at room temperature, or into RNA later -ICE solution, if frozen. The solution permeates the cells and stabilizes the RNA. The samples are then stored at 4°C using RNA later  reagent, or at –20°C when using RNA later -ICE RNA Stabilization Solution. Samples can be shipped on wet ice or even at room temperature if shipped overnight.

28. Rna preparation 4 general techniques: organic extraction methods spin basket formats magnetic particle methods direct lysis methods

29. Organic extraction method Gold standard for RNA preparation. Sample is homogenized in a phenol-containing solution and then centrifuged. During centrifugation, the sample separates into three phases: a lower organic phase, a middle phase that contains denatured proteins and gDNA, and an upper aqueous phase that contains RNA. The upper aqueous phase is recovered and RNA is collected by alcohol precipitation and rehydration. Benefit Drawbacks Rapid denaturation of nucleases and stabilization of RNA The use and associated waste of chlorinated organic reagents Scalable format Laborious and manually intensive processing Difficult to automate

30. Filter based, spin basket format Filter-based, spin basket formats utilize membranes (usually glass fiber, derivatized silica, or ion exchange membranes) that are seated at the bottom of a small plastic basket.  Samples are lysed in a buffer that contains RNase inhibitors (usually guanidine salts), and nucleic acids are bound to the membrane by passing the lysate through the membrane using centrifugal force. Wash solutions are subsequently passed through the membrane and discarded. An appropriate elution solution is applied and the sample is collected into a tube by centrifugation. Some formats can be processed by either centrifugation or vacuum using specialized manifolds. Benefit Drawbacks Convenience and ease of use Propensity to clog with particulate material Amenable to single-sample and 96-well processing Retention of large nucleic acids such as gDNA Ability to automate Fixed binding capacity within a manufactured format Ability to manufacture membranes of various dimensions When automated, requirements for complex vacuum systems or centrifugation

31. Magnetic particle method Utilize small (0.5–1 µm) particles that contain a paramagnetic core and surrounding shell modified to bind to entities of interest. Paramagnetic particles migrate when exposed to a magnetic field, but retain minimal magnetic memory once the field is removed  allows the particles to interact with molecules of interest based on their surface modifications, be collected rapidly using an external magnetic field, and then be resuspended easily once the field is removed. Samples are lysed in a solution containing RNase inhibitors and allowed to bind to magnetic particles. The magnetic particles and associated cargo are collected by applying a magnetic field. After several rounds of release, resuspension in wash solutions, and recapture, the RNA is released into an elution solution and the particles are removed. Benefits Drawbacks No risk of filter clogging Potential carry-through of magnetic particles into eluted samples Solution-based binding kinetics increase the efficiency of target capture Slow migration of magnetic particles in viscous solutions The magnetic format allows for rapid collection/ concentration of sample Capture/release of particles can be laborious when performed manually Increased ease of implementation on instrument platforms Ability to automate Wide availability of surface chemistries

32. Direct lysis method U tilizing lysis buffer formulations that disrupt samples, stabilize nucleic acids, and are compatible with downstream analysis. Typically, a sample is mixed with lysis agent, incubated for some amount of time under specified conditions, and then used directly for downstream analysis. If desired, samples can often be purified from stabilized lysates. By eliminating the need to bind and elute from solid surfaces, direct lysis methods can avoid bias and recovery efficiency effects that may occur when using other purification methods. Benefits of direct lysis methods Extremely fast and easy Highest potential for accurate RNA representation Can work well with very small samples Amenable to simple automation Scalable Drawbacks of direct lysis methods Inability to perform traditional analytical methods such as spectrophotometric measurement of yield Dilution-based (most useful with concentrated samples) Potential for suboptimal performance unless developed/optimized with downstream analysis Potential for residual RNase activity if lysates are not handled properly

33. STORAGE OF ISOLATED RNA The last step in every RNA isolation protocol, whether for total or mRNA preparation, is to resuspend the purified RNA pellet. After painstakingly preparing an RNA sample, it is crucial that RNA be suspended and stored in a safe, RNase-free environment. We recommend storing RNA at –80°C in single-use aliquots, resuspended in one of several RNA storage solutions designed for this purpose: THE RNA Storage Solution (1 mM sodium citrate, pH 6.4 ± 0.2) 0.1 mM EDTA (in DEPC-treated ultrapure water) TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0) RNA secure  Resuspension Solution TE and 0.1 mM EDTA solutions are often specified in common RNA isolation and analysis protocols.

34. How to extract RNA from tissue? Collect the tissue sample and immediately snap-freeze it in liquid nitrogen or store it at –80°C for preservation. Grind the tissue sample to a fine powder using a pre-chilled mortar and pestle or homogenizer. Add Trizol or another RNA extraction reagent to the tube containing the tissue sample, following the recommended volume based on the sample size. Vortex vigorously to lyse the cells. Incubate the tube at room temperature for 5 minutes. Add chloroform to the tube in an equal volume. Shake vigorously for 15 seconds. Centrifuge the tube at high speed (12,000–16,000 x g) for 15 minutes at 4°C. Transfer the upper aqueous phase to a fresh tube, avoiding the interphase and organic phase. Add an equal volume of isopropanol to the tube and mix gently. Incubate at room temperature for 10 minutes. Centrifuge at high speed for 10 minutes at 4°C to pellet the RNA. Remove the supernatant, wash the RNA pellet with 75% ethanol, and centrifuge again for 5 minutes at 4°C.  Remove the ethanol, air-dry the RNA pellet briefly, and dissolve it in RNase-free water or an appropriate buffer. Option to treat the RNA sample at this step with DNase I to remove genomic DNA contamination. Add an RNase inhibitor to protect the RNA from degradation and store the RNA sample at –80°C or use it for downstream applications.

35. Next-generation sequencing: 3 main challenges (& solutions!) in NGS sample preparation Getting reliable data in next-generation sequencing (NGS) is all about the DNA (or RNA) you put in. DNA for sequencing might come from a variety of sources, including fresh tissue, formalin-fixed paraffin-embedded (FFPE) tissue, cultured cells, and liquid biopsies. Each source comes with its own challenges for maximizing the three key aspects: Quantity Integrity Purity

36. Challenge #1: Yield Different workflows and kits vary significantly in the amount of starting material required. Your workflow might require you to use a specific type of kit, and therefore starting DNA, or vice versa. It’s important to understand which workflows and kits suit your application and the typical amounts of starting material they need. Your fragmentation method can also affect your final DNA yield. Physical fragmentation can result in unexpectedly small DNA fragments which can be lost, reducing the amount of DNA available for sequencing. If you have the option, enzymatic fragmentation can provide better predictability and control over fragmentation.

37. Challenge #2: integrity Having enough DNA won’t make for accurate sequencing if your DNA is degraded. Degradation can affect all kinds of samples, especially long-term storage and exposure to fixatives (FFPE samples) A DNA integrity number (DIN) measurement can indicate the level of DNA damage. DIN measurement is an easy method to check DNA integrity. Extracting a little more DNA can compensate for low quality to some extent. If you can’t acquire better samples, DNA repair might improve your outcomes. Several commercial kits can, for example, modify blocked 3’ ends or fix DNA nicks. These simple repairs help make more fragments suitable for sequencing.

38. Challenge #3: impurities Producing reliable results in sequencing requires samples free of proteins, organic solvents and surfactants. Measure DNA purity by looking at the 260:280 nm absorbance ratio. A high-purity sample should have a 260:280 ratio of 1.8 to 2.0. Nucleic acids have an absorbance maximum at 260 nm and finding a ratio below 1.8 can indicate contamination. As a secondary check, measure the 260:230 ratio, which will detect the presence of commonly used solvents and surfactants, such as phenol and EDTA. Values between 2.0 and 2.2 indicate high purity. Remove hemoglobin by preferential lysis of red blood cells early in your workflow. Remove heparin by washing. Do a phenol–chloroform extraction to reduce protein contamination. Use a  phenol-free extraction kit  to remove phenol contamination.

39. Fundamentals of NGS sample preparation Steps include: DNA extraction A mplification L ibrary preparation Selection or purification Quality control

40. Next-generation sequencing methodology. (Reprinted with permission from Nayarisseri A, Yadav M, Bhatia M, et al.: Impact of next-generation whole-exome sequencing in molecular diagnostics, Drug Invention Today 5:327–334, 2013.)

41. Workflow and data analysis of targeted sequencing by next-generation sequencing platforms for clinical applications.

42. Dna extraction The first step in every sample prep protocol is extracting the genetic material– DNA or RNA– from cells and tissues. Other molecules, such as RNA and proteins, interfere with the sequencing process and must be removed before doing anything else. The specific tissue type and storage conditions determine the details of this extraction process. Extraction entails breaking down the extracellular matrix and opening the cell membranes using enzymes, solvents, or surfactants. The DNA in the resulting mixture must then be isolated. The traditional gold standard in DNA isolation is phenol-based extraction. Phenol is a hydrophobic solvent that denatures and dissolves proteins, removing them from the DNA-containing aqueous phase. However, it can be tricky to work with, and users need to be careful not to contaminate the aqueous phase with phenol. Spin columns that specifically bind DNA provide an alternative and are an easy-to-use, but more expensive, method to wash away the debris. Chloroform-based extraction, another alternative, enables you to isolate high-quality DNA without phenol, and commercial kits can include a resin that minimizes the risk of contamination.

43. Amplification methods Amplification after extraction is optional, depending on your application and sample size. For example, whole genome sequencing (WGA) with 2 µg of starting material does not necessarily require further amplification. But, with nanograms—or even picograms—of starting material, amplification becomes essential to obtain sufficient coverage for reliable sequence calls. Isothermal amplification and polymerase chain reaction (PCR) are two common methods to increase the amount of input DNA. PCR uses generic primers to amplify the starting material in a highly uniform manner, but tends to be more error-prone than multiple displacement amplification (MDA). MDA is an isothermal method, often based on Phi29 polymerase, and excels in accuracy with low rates of false-positives and false-negatives. MDA’s main drawback is overrepresentation of some regions of the genome.

44. Library preparation Most NGS platforms analyze DNA in uniform, bite-size pieces, created by DNA fragmentation. This process generates a ‘library’ of fragments with a narrow length distribution that is optimal for the sequencing platform. DNA fragmentation Both mechanical fragmentation (shearing) and enzymatic methods are suitable for NGS. Mechanical methods enable random shearing to produce a variety of overlapping fragments for any given region of the genome. This is ideal for de novo assembly. Enzymatic methods are relatively fast and require less investment upfront but have some ‘bias’, cleaving some sites preferentially, making de novo assembly more challenging without the variety of overlapping fragments.

45. DNA end-repair The fragments generated have single-stranded, ‘sticky’ ends. The next step, end-repair, fills in these sticky ends to create blunt ends, ready for adaptor ligation. Adaptors Adaptors are then bound to both the 5’ and the 3’ ends of the library fragments. They are specific to the sequencing platform, but ultimately all serve to enable in-platform clonal amplification, i.e. Illumina’s bridge amplification or BGI’s rolling circle amplification. The adaptors are designed to bind to the sequencer-specific substrate, such as a patterned flow cell, contain sequences to enable amplification, and can have barcodes for fragment identification.

46. Targeted sequencing In amplicon-based target enrichment, the fragmentation and end-repair steps tend to be unnecessary. Pulling the targeted regions out as amplicon fragments with blunt ends enables you to go directly to adaptor ligation. Hybridization-based enrichment does require fragmentation. The hybridization probes pull out the regions of interest from the library of overlapping fragments, ready for end-repair.

47. Dna sequencing: size selection and purification It might be necessary to ‘clean up’ your library before sequencing by removing fragments that won’t produce relevant data. For NGS workflows that have narrow size requirements, discarding fragments that are either too large or too small to produce useful results can improve sequencing efficiency. There are different protocols for size selection, which might involve gel electrophoresis or magnetic bead-based selection. Magnetic beads also provide a quick and easy method for final clean-up.

48. DNA quality control A final step before proceeding to sequencing is to confirm the quality and quantity of the DNA. Both parameters contribute to the confidence in your sequencing data. You can measure the quantity of your DNA using fluorescence- or qPCR-based methods. For qualitative validation, many protocols use the Agilent TapeStation ™ or Bioanalyzer™.

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