Services
BioSistemika offers a range of contract services in the field of nucleic acid analyses (DNA, RNA).
Highly skilled and professional team in laboratories where ISO 17025 and ISO 9001 accredited methods are being carried out will ensure high quality quantitative and qualitative analysis of nucleic acids with real-time PCR in various types of biological samples (mammalian, insect, plant, bacterial, viral, etc) including validation of microarray gene expression data, coaching and counselling. For further information please contact us at info@biosistemika.com.
The team has more than 40 publications in international scientific journals and books chapters from all fields of services (real-time PCR, DNA microarrays, bioinformatics and quality assurance) ensuring high quality level of knowledge, experience and consequently services.
We cover complete workflow (wet-lab and dry-lab) including coaching & counselling on all steps:
Experiment design [back to top]
Background & Applications
Have you ever heard of the expression "garbage in, garbage out"? An important step that has to happen before lab work in order to prevent that situation is experiment design. Usually it does not receive enough attention or is simply omitted.
Experiment design begins with asking yourself a basic question "What do I expect from the experiment?". The next step is to design the experiment in a way that you will be able to answer those question(s). This means that all the right combinations of controls and treatments need to be set up, the right number of samples, technical and biological replicates for each comparison, etc. In the end analysis of the data will cause less problems. It is advisable to involve a statistician or a bioinformatician in this step.
We offer
We offer general coaching and counselling on experiment design from the experimentalist's (e.g. biological) point of view as well as from statistical and bioinformatic point of view.
Sample preparation [back to top]
Background & Applications
Sample preparation begins at the point when the biological material is collected (e.g. drawing patient's blood, cutting a leaf from potato, putting an insect into the freezer, cutting of a piece of hair, etc…).
It usually consists of homogenisation, nucleic acid isolation, purification and storage. Each of these steps can be more or less complex, depending on the biological material and its quantity. In case of RNA (intended for gene expression profiling) time from sample collection until its stabilisation in a storage or RNA extraction buffer is of crucial importance. It needs to be short.
In case of plant material special care needs to be taken to avoid co-isolation of inhibitors of PCR reaction.
Methods and platforms we cover
• Homogenisation, stabilisation and storage of samples
• DNA/RNA extraction from mammal tissues and cell cultures including single-cells, plant tissues, cells; using kits or conventional approaches
• DNA/RNA quality control and quantification (see below)
RNA/DNA Quality control [back to top]
Background & Applications
RNA quality control has become an essential step in the gene expression workflow. Different approaches enable not only the quality control (check whether RNA is intact or degraded) but also quantification of the RNA and check for contamination with proteins and genomic DNA. Only the RNA of the highest quality will give reliable end results.
Methods and platforms we cover
• Gel Electrophoresis (Ethidium bromide or SybrSafe staining) - integrity check
• UV/VIS spectroscopy (Nanodrop) - integrity check and quantification
• RNA LabChip® Analysis (Agilent BioAnalyzer)- integrity check and quantification
Reverse transcription and (pre)amplification [back to top]
Background & Applications
Reverse transcription is an essential step in gene expression analysis workflow, transcribing mRNA into double-stranded complementary DNA (cDNA). It is also recommended that RNA samples are treated with DNase to remove any genomic DNA that is co-isolated with RNA prior reverse transcription.
In case of low quantities of RNA (e.g. small samples, single cell analysis) there is the option to amplify (preamplify) cDNA before PCR/qPCR to enhance PCR/qPCR detection.
Methods and platforms we cover
• DNase treatment with DNase I enzyme
• reverse transcription using different kits
• preamplification using different kits (isothermal or PCR-based amplifications)
Gene expression [back to top]
Background & Applications
Analysis of transcriptome (gene expression) or transcription profiling is one of the most commonly used molecular biology techniques nowadays. It is used in basic and applied research in various fields of life sciences such as study of complex networks of genes interaction in specific processes or regulatory pathways in drug discovery, diagnostics of disease and so on.
Currently two predominating techniques are used, qPCR and DNA-microarrays, both covered by our services. In both cases samples are prepared in much the same way: RNA is isolated, quality checked and reverse transcribed. In case of DNA-microarray additional steps of RNA/cDNA labelling with fluorescent dyes and sometimes simultaneous amplification is performed prior to hybridisation to microarrays.
New techniques are already on the horizon such as massive parallel sequencing (new generation sequencing) and high-density qPCR technologies (e.g. Fluidigm). They still require some time to establish themselves in the research community.
Methods and platforms we cover
• TaqMan®
• Sybr Green I
• commercial low density qPCR arrays
• high density oligonucleic DNA-microarrays
Platforms:
• qPCR: Applied Biosystems 7900 HT, Roche Light cycler 480, Smartcycler, Fluidigm (soon to come)
• DNA-microarrays: Illumina, Agilent, Affymetrix, custom arrays
Bioinformatics and data analysis [back to top]
Background & Applications
Bioinformatics helps experimentalists in designing their experiments so that they follow statistical guidelines and also perform the data analysis on experimental data. One of the last steps of bioinformatics includes visualising results in ways that ease the interpretation of the data for experimentalists. It includes both statistical and data mining approaches.
We cover
• gene expression studies performed with real-time PCR, including large scale gene expression studies (several hundreds of samples screened with 10 or more genes)
• microarray gene expression studies (platforms: Illumina, Affymetrix, Agilent, custom oligonucleotide single/dual label microarray platforms)
We perform following analyses on gene expression DNA microarray data
• data import, normalisation, filtering, quality control
• search for differentially expressed genes using linear models
• clustering, pathway analysis, gene ontology analysis,
• deposition of microarray gene expression studies data into public databases (Gene Expression Omnibus (GEO), ArrayExpress)
Molecular diagnostics methods validation [back to top]
Background & Applications
Real-time PCR has been around for approximately 15 years. In this time reliable instruments (platforms) and chemistries have been developed enabling slow but reliable penetration of real-time PCR into diagnostic applications, such as medical diagnostics.
As soon as a diagnostic laboratory is required to comply with quality standards (e.g. GLP, ISO 17025) the "old" methods need to be validated and the new methods developed according to quality standards. That on its own is a valuable know-how and is sometimes very challenging for laboratories that are doing it for the first time or simply do not have human resources for it. Here is where we step in.
Methods and platforms we cover
• guidelines for validation and validation of your existing real-time PCR methods according to GLP (good laboratory practice) or ISO 17025
• preparation of all required documentation (protocols, standard operating procedures, reports) tailored around your quality assurance requirements
This is possible for real-time PCR diagnostic qualitative and quantitative methods based on TaqMan® and Sybr Green I chemistry and Applied Biosystems 7900 HT and Roche Light cycler 480 real-time PCR instruments.
Feasibility studies [back to top]
Are you thinking of switching to real-time PCR method? Would you like to know if that is possible for your specific application and how much a routine analysis would cost you compared to your conventional method?
We can provide an insight into technical and financial feasibility of the project:
• answer the question whether detection of your target organism (e.g. bacteria) is possible with real-time PCR
• search for existing or design of customised amplicon or set of amplicons
• set-up analysis containing selected amplicons and all required controls
• validate the analysis
• prepare standard operating protocols for routine analysis
• educate the laboratory personnel
• estimate the costs for all phases of the project:
- implementation of the analysis into the laboratory
- validation of the analysis
- cost per sample in case of routine analysis
Quality assurance [back to top]
Background & Applications
Quality assurance is a wide term describing activities and measures that actually make the quality system in a laboratory, department or an institution. It is much more than just a set of rules, standards, operating procedures, and guidelines, it is a way of working and thinking. Once that is understood and implemented, quality assurance stops being a hurdle and starts being an advantage.
Our aim is to help with our expert knowledge to build slim and efficient quality assurance systems in molecular biology based diagnostic (testing) laboratories around your quality assurance requirements in your regulated environment, making all workflows traceable and documented according to ISO 17025 and ISO 9001. Both standards have a very similar "core".
We cover
• guidelines for ensuring general traceability of workflows (e.g. recieving samples, processing samples, isolation of nucleic acids, performing qPCR analysis, collecting data, analysing data, dealing with unceartanty of measurments, reporting, sending out reports/results, etc.)
• sample and data handling in a traceable way
• preparation of Standard operating procedures (SOP) and protocols
• on site validation of methods or coaching (see above)
• laboratory organisation, equipment management and calibration (organising sample flows, avoiding contaminations, guidelines for external and internal periodic equipment validations/calibrations)
• coaching for internal and external audits
• coaching and handling of corrective and preventive actions
• setting-up documentation management system and general traceability of documents
Molecular Laboratory setup [back to top]
Background & Applications
In order to reduce cross contamination especially in diagnostic laboratories that are accredited under quality standards it is advised that the sample workflow is one way only and divided into separated steps. This means that the sample homogenisation is separated from nucleic acid extraction etc. This should also be reflected in laboratory organisation and workflow. Since there are many different instruments available for different purposes of each step of the workflow, it is difficult to decide which to choose.
We offer
With years of experience with molecular diagnostic laboratory ISO 17025 accreditation using qPCR, we can advise you on buying the most suitable equipment for your purpose and help you to organise the workflow in laboratory to prevent contamination.
Highly skilled and professional team in laboratories where ISO 17025 and ISO 9001 accredited methods are being carried out will ensure high quality quantitative and qualitative analysis of nucleic acids with real-time PCR in various types of biological samples (mammalian, insect, plant, bacterial, viral, etc) including validation of microarray gene expression data, coaching and counselling. For further information please contact us at info@biosistemika.com.
The team has more than 40 publications in international scientific journals and books chapters from all fields of services (real-time PCR, DNA microarrays, bioinformatics and quality assurance) ensuring high quality level of knowledge, experience and consequently services.
Something _________ in your work? We have it!
Reliable. Professional. BioSistemika.
Reliable. Professional. BioSistemika.
We cover complete workflow (wet-lab and dry-lab) including coaching & counselling on all steps:
- Experiment design ... more »
- Sample preparation ... more »
- RNA/DNA Quality control ... more »
- Reverse transcription and (pre)amplification ... more »
- Gene expression ... more »
- Bioinformatics and data analysis ... more »
- Molecular diagnostics methods validation ... more »
- Feasibility studies ... more »
- Quality assurance ... more »
- Lab setup ... more »
Experiment design [back to top]
Background & Applications
Have you ever heard of the expression "garbage in, garbage out"? An important step that has to happen before lab work in order to prevent that situation is experiment design. Usually it does not receive enough attention or is simply omitted.
Experiment design begins with asking yourself a basic question "What do I expect from the experiment?". The next step is to design the experiment in a way that you will be able to answer those question(s). This means that all the right combinations of controls and treatments need to be set up, the right number of samples, technical and biological replicates for each comparison, etc. In the end analysis of the data will cause less problems. It is advisable to involve a statistician or a bioinformatician in this step.
We offer
We offer general coaching and counselling on experiment design from the experimentalist's (e.g. biological) point of view as well as from statistical and bioinformatic point of view.
Sample preparation [back to top]
Background & Applications
Sample preparation begins at the point when the biological material is collected (e.g. drawing patient's blood, cutting a leaf from potato, putting an insect into the freezer, cutting of a piece of hair, etc…).
It usually consists of homogenisation, nucleic acid isolation, purification and storage. Each of these steps can be more or less complex, depending on the biological material and its quantity. In case of RNA (intended for gene expression profiling) time from sample collection until its stabilisation in a storage or RNA extraction buffer is of crucial importance. It needs to be short.
In case of plant material special care needs to be taken to avoid co-isolation of inhibitors of PCR reaction.
Methods and platforms we cover
• Homogenisation, stabilisation and storage of samples
• DNA/RNA extraction from mammal tissues and cell cultures including single-cells, plant tissues, cells; using kits or conventional approaches
• DNA/RNA quality control and quantification (see below)
RNA/DNA Quality control [back to top]
Background & Applications
RNA quality control has become an essential step in the gene expression workflow. Different approaches enable not only the quality control (check whether RNA is intact or degraded) but also quantification of the RNA and check for contamination with proteins and genomic DNA. Only the RNA of the highest quality will give reliable end results.
Methods and platforms we cover
• Gel Electrophoresis (Ethidium bromide or SybrSafe staining) - integrity check
• UV/VIS spectroscopy (Nanodrop) - integrity check and quantification
• RNA LabChip® Analysis (Agilent BioAnalyzer)- integrity check and quantification
Reverse transcription and (pre)amplification [back to top]
Background & Applications
Reverse transcription is an essential step in gene expression analysis workflow, transcribing mRNA into double-stranded complementary DNA (cDNA). It is also recommended that RNA samples are treated with DNase to remove any genomic DNA that is co-isolated with RNA prior reverse transcription.
In case of low quantities of RNA (e.g. small samples, single cell analysis) there is the option to amplify (preamplify) cDNA before PCR/qPCR to enhance PCR/qPCR detection.
Methods and platforms we cover
• DNase treatment with DNase I enzyme
• reverse transcription using different kits
• preamplification using different kits (isothermal or PCR-based amplifications)
Gene expression [back to top]
Background & Applications
Analysis of transcriptome (gene expression) or transcription profiling is one of the most commonly used molecular biology techniques nowadays. It is used in basic and applied research in various fields of life sciences such as study of complex networks of genes interaction in specific processes or regulatory pathways in drug discovery, diagnostics of disease and so on.
Currently two predominating techniques are used, qPCR and DNA-microarrays, both covered by our services. In both cases samples are prepared in much the same way: RNA is isolated, quality checked and reverse transcribed. In case of DNA-microarray additional steps of RNA/cDNA labelling with fluorescent dyes and sometimes simultaneous amplification is performed prior to hybridisation to microarrays.
New techniques are already on the horizon such as massive parallel sequencing (new generation sequencing) and high-density qPCR technologies (e.g. Fluidigm). They still require some time to establish themselves in the research community.
Methods and platforms we cover
• TaqMan®
• Sybr Green I
• commercial low density qPCR arrays
• high density oligonucleic DNA-microarrays
Platforms:
• qPCR: Applied Biosystems 7900 HT, Roche Light cycler 480, Smartcycler, Fluidigm (soon to come)
• DNA-microarrays: Illumina, Agilent, Affymetrix, custom arrays
Bioinformatics and data analysis [back to top]
Background & Applications
Bioinformatics helps experimentalists in designing their experiments so that they follow statistical guidelines and also perform the data analysis on experimental data. One of the last steps of bioinformatics includes visualising results in ways that ease the interpretation of the data for experimentalists. It includes both statistical and data mining approaches.
We cover
• gene expression studies performed with real-time PCR, including large scale gene expression studies (several hundreds of samples screened with 10 or more genes)
• microarray gene expression studies (platforms: Illumina, Affymetrix, Agilent, custom oligonucleotide single/dual label microarray platforms)
We perform following analyses on gene expression DNA microarray data
• data import, normalisation, filtering, quality control
• search for differentially expressed genes using linear models
• clustering, pathway analysis, gene ontology analysis,
• deposition of microarray gene expression studies data into public databases (Gene Expression Omnibus (GEO), ArrayExpress)
Molecular diagnostics methods validation [back to top]
Background & Applications
Real-time PCR has been around for approximately 15 years. In this time reliable instruments (platforms) and chemistries have been developed enabling slow but reliable penetration of real-time PCR into diagnostic applications, such as medical diagnostics.
As soon as a diagnostic laboratory is required to comply with quality standards (e.g. GLP, ISO 17025) the "old" methods need to be validated and the new methods developed according to quality standards. That on its own is a valuable know-how and is sometimes very challenging for laboratories that are doing it for the first time or simply do not have human resources for it. Here is where we step in.
Methods and platforms we cover
• guidelines for validation and validation of your existing real-time PCR methods according to GLP (good laboratory practice) or ISO 17025
• preparation of all required documentation (protocols, standard operating procedures, reports) tailored around your quality assurance requirements
This is possible for real-time PCR diagnostic qualitative and quantitative methods based on TaqMan® and Sybr Green I chemistry and Applied Biosystems 7900 HT and Roche Light cycler 480 real-time PCR instruments.
Feasibility studies [back to top]
Are you thinking of switching to real-time PCR method? Would you like to know if that is possible for your specific application and how much a routine analysis would cost you compared to your conventional method?
We can provide an insight into technical and financial feasibility of the project:
• answer the question whether detection of your target organism (e.g. bacteria) is possible with real-time PCR
• search for existing or design of customised amplicon or set of amplicons
• set-up analysis containing selected amplicons and all required controls
• validate the analysis
• prepare standard operating protocols for routine analysis
• educate the laboratory personnel
• estimate the costs for all phases of the project:
- implementation of the analysis into the laboratory
- validation of the analysis
- cost per sample in case of routine analysis
Quality assurance [back to top]
Background & Applications
Quality assurance is a wide term describing activities and measures that actually make the quality system in a laboratory, department or an institution. It is much more than just a set of rules, standards, operating procedures, and guidelines, it is a way of working and thinking. Once that is understood and implemented, quality assurance stops being a hurdle and starts being an advantage.
Our aim is to help with our expert knowledge to build slim and efficient quality assurance systems in molecular biology based diagnostic (testing) laboratories around your quality assurance requirements in your regulated environment, making all workflows traceable and documented according to ISO 17025 and ISO 9001. Both standards have a very similar "core".
We cover
• guidelines for ensuring general traceability of workflows (e.g. recieving samples, processing samples, isolation of nucleic acids, performing qPCR analysis, collecting data, analysing data, dealing with unceartanty of measurments, reporting, sending out reports/results, etc.)
• sample and data handling in a traceable way
• preparation of Standard operating procedures (SOP) and protocols
• on site validation of methods or coaching (see above)
• laboratory organisation, equipment management and calibration (organising sample flows, avoiding contaminations, guidelines for external and internal periodic equipment validations/calibrations)
• coaching for internal and external audits
• coaching and handling of corrective and preventive actions
• setting-up documentation management system and general traceability of documents
Molecular Laboratory setup [back to top]
Background & Applications
In order to reduce cross contamination especially in diagnostic laboratories that are accredited under quality standards it is advised that the sample workflow is one way only and divided into separated steps. This means that the sample homogenisation is separated from nucleic acid extraction etc. This should also be reflected in laboratory organisation and workflow. Since there are many different instruments available for different purposes of each step of the workflow, it is difficult to decide which to choose.
We offer
With years of experience with molecular diagnostic laboratory ISO 17025 accreditation using qPCR, we can advise you on buying the most suitable equipment for your purpose and help you to organise the workflow in laboratory to prevent contamination.