IMS Microarray Core facility (IMCF)- Protocols
Up to date manuals, including the CustomSeq™ resequencing array protocol and design guide (sample preparation and design of resequencing arrays), E.coli antisense array protocol (E.coli antisense expression arrays), Expression analysis technical manual (Eukaryotic and Prokaryotic expression array protocols), the Mapping assay manual (10K SNP array protocols) and a Custom array design guide (for designing custom made gene expression arrays) are available from the Affymetrix website. The Affymetrix technical manuals provide detailed information on protocols, suppliers and reagents. Please follow these protocols and use the recommended reagents/suppliers, as these have been developed and validated specifically for Affymetrix GeneChip™ arrays. Although the sample preparation costs are high, unfortunately a number of cheaper reagents from alternative suppliers have proved to be of insufficient quality for preparation of labelled-cRNA from total RNA, so trying out alternative reagents may be more expensive in the long run.
Sample Preparation for GeneChip™ Expression Arrays
Please read the notes on experimental design before you begin your study. Analysis of gene expression on GeneChip™ arrays typically involves extraction of total RNA from the sample of interest, clean-up of the total RNA, reverse transcription of total RNA to cDNA, clean-up of cDNA, in vitro transcription of cDNA to cRNA using biotin-dUTP and biotin-dCTP to label the cRNA, fragmentation of biotin-labelled cRNA (~35 -200bp to improve hybridisation) and addition of fragmented biotin-labelled cRNA to a hybridisation cocktail (contains buffer, blocking reagents and controls), prior to hybridisation to the GeneChip™ array. I have put together a few technical tips on sample preparation for GeneChip™ expression arrays, these should be read in conjunction with the Affymetrix expression analysis technical manual. Once you have identified your 'interesting gene set', validation of a subset of your gene expression changes is recommended.
Poly-A spike controls can be added to total RNA or mRNA preparations to monitor efficiency of target preparation, hybridisation, washing and staining. Bacterial clones containing the relevant recombinant plasmids can be obtained from ATCC and prepared according to the instructions in the Expression analysis technical manual. Alternatively, a Eukaryotic poly A RNA control kit can be purchased directly from Affymetrix. This kit, which contains exogenous pre-mixed prokaryotic polyA controls sufficient for 100 reactions, can be used in either the standard or the small sample target labelling assays and it provides a sensitive indication of the labelling efficiency independent of starting sample quality.
Preparation of total RNA, cDNA and cRNA from cells or tissues: Trizol preparation of total RNA from cells or tissues provides high yield, good quality RNA that is suitable for use in the preparation of cDNA. A detailed protocol for preparation of total RNA using Trizol, along with tips for working with RNA, is provided with the reagent.
The Affymetrix One Cycle Target labelling kit is used to reverse transcribe total (or mRNA) into cDNA and the Affymetrix IVT labelling kit is used to amplify cDNA into biotin-labelled cRNA, for hybridisation to Affymetrix GeneChip™ arrays. The One Cycle cDNA and IVT kits can be purchased with appropriate control reagents as a single kit. The Affymetrix expression analysis technical manual provides detailed protocols for synthesis and clean-up (see below) of cDNA and biotin-labelled cRNA. It is important that accurate equipment is used for heating/cooling steps in cDNA and cRNA synthesis - it is preferable to use a thermal cycler or a cool block for heating or cooling, respectively. In our hands, these protocols produce higher yields of labelled cRNA than the previous versions outlined below.
The following links provide the original protocols for synthesis of cDNA by reverse transcription and in vitro transcription of biotin-labelled cRNA. Ongoing projects utilising these methods should not transfer to the new protocols outlined aboved. Modification of sample preparation protocols within a study can increase systemic variation in your data.
Preparation of total RNA from blood: Preparation of total RNA from blood can be difficult due to problems with activating the cells during blood collection and high RNase activity in this 'tissue'. Affymetrix have released a technical note assessing and comparing total RNA/cRNA quantity/quality and cellular activation using a number of common methods for collection and isolation of RNA from blood. An appendix of signature genes for different cellular sub-populations within various blood fractions is also available. Affymetrix have also developed a globin reduction protocol to reduce the globin mRNA population in total RNA, with the aim of improving the sensitivity of gene expression analysis when using whole, rather than fractionated, blood for total RNA isolation.
T7-Oligo(dT) Primer: It is essential to use a high quality primer for synthesis of cDNA and cRNA. This is a long oligonucleotide which degrades easily. You should use a trusted supplier to order HPLC purified primer - please note that some users have had problems obtaining good quality T7-Oligo(dT) primer from the suppliers they use for their PCR primers. Affymetrix previously recommended GenSet. The core facility uses Sigma-Genosys and obtains good quality cRNA (this is the only reagent where we are not using the recommended supplier). Affymetrix supply a T7-Oligo(dT) Promoter Primer Kit, which provides sufficient primer for 150 reactions; ordering and technical information are available on this product. This is more expensive than buying in your own primer but may be a preferable option if you are having problems with sample preparation, as this primer has been QC'd against GeneChip™ arrays. Your stock and working concentration of T7-Oligo(dT) primer should be aliquoted upon receipt (prior to freezing the primer at -80oC), to limit the number of freeze-thaw cycles to two.
Sample Clean-up: Affymetrix supply a sample clean-up module kit containing sufficient reagents to clean 30 cDNA preparations, 30 cRNA preparations and fragment 30 cRNA preparations. This kit is now recommended for these steps in the protocol as it is less time consuming and is approximately the same cost as the old clean-up protocols. However, in our hands, higher yields are obtained with the previous protocols (RNeasy kit, Qiagen for total RNA clean-up and cRNA clean-up and phenol/ ethanol precipation for cDNA clean-up).
Small sample preparations: The one-cycle protocols require a minimum of 1-5mg of total RNA to provide sufficient labelled cRNA for hybridisation to GeneChip™ expression arrays. For some studies, it is not possible to obtain sufficient material to extract this amount of total RNA. The Affymetrix two cycle target labelling assay provides protocols for the preparation of biotin-labelled cRNA from as little as 10-100ng of total RNA. If it is necessary for you to use this protocol, it is essential to validate expression analysis on targets prepared in this way. The small scale protocol involves 2 rounds of amplification, thereby increasing the potential for non-linear amplification of the original material which could result in over-or under-representation of some transcripts (e.g. high or low expressed transcripts, respectively). One option to avoid small scale preparations is to pool tissue samples (e.g. multiple biopsies from the same patient, multiple specimens from the same animal or litter - for example, multiple lymph nodes from a single mouse or multiple brains from a litter of rats). Pooling requires homogeneous populations and good experimental design to avoid deleterious effects of outliers and amplification of small scale material requires robust validation prior to full scale gene expression analysis- advice should be obtained prior to embarking on either of these routes. Modified and alternative small scale preparation protocols are available from Elaina Collie-Duguid.
Hybridisation cocktail:
If you intend to run one Test3 array and one full scale Gene Expression array from your sample, please follow the instructions for preparing a hybridisation cocktail for '49 Format (Standard)/ 69 Format array' (Table 2.3.1, page 2.3.7 Affymetrix expression analysis technical manual), which will provide sufficient volume/cRNA (300ml/15mg) to hybridise your sample to these 2 arrays.
The hybridisation controls are provided in the Eukaryotic Hybridisation Control kit which can be ordered directly from Affymetrix. The controls are pre-mixed at staggered concentrations (1.5-100pM) and are ready to add directly to the hybridisation cocktail. These allow monitoring of the hybridisation process for trouble shooting and allow comparison with house-keeping genes in the assessment of sample quality. Control oligoB2, used by the software for alignment of the grid, is also provided in this kit.
Please 0.2mm filter the hybridisation buffer to reduce background on the arrays.
Sample Evaluation: It is advisable to monitor the quality and yield of your sample at each step of the protocol (i.e. total RNA, cDNA, labelled-cRNA and fragmented labelled-cRNA). These can be assessed on an agorose gel or using an Agilent Bioanalyser (this system is available at The Rowett Research Institute, Aberdeen: Samples can be run in batches of 12 at a cost of £30+VAT/plate. The contact for this service is Gillian Campbell). Due to the expense and time associated with sample preparation as well as the large amount of data produced in a microarray experiment, it is advisable to keep a detailed log of each sample to facilitate QC, storage and retrieval of sample preparations. The various fractions produced during preparation of the sample for hybridisation to GeneChip™ arrays are stable and can be stored long-term at -80oC for future analyses (e.g. analysis on other GeneChip™ arrays or in validation studies).
Troubleshooting: The most common sources of problems with sample preparation are poor quality or insufficient RNA, poor quality T7-oligo(dT) primer for cDNA and cRNA synthesis or inaccurate equipment for heating/cooling steps in cDNA/cRNA synthesis.