GENERAL GENOMIC SERVICE - GENE EXPRESSION UNIT

1. To request the services of the Gene Expression Unit, its technical personnel must first be contacted in order to inform them of the type of service being requested and the number and type of samples to be analysed.
2. In the case of external users, the acceptance of the budget has to be sent signed, previous to the service request. In the case of internal users (UPV/EHU) the signature of acceptance of the budget is only required for the services of RNA isolation, microarrays and Q-PCR. The invoicing data or information must be sent in advance for the acceptance of the realization of the service.
3. Once the technical personnel of the Gene Expression Unit confirmed the acceptance of the service by email, the user requests the service by completing the request form for the relevant service, which will be handed over together with the samples to be analysed.  The samples must be delivered in individual Eppendorf tubes and duly labelled with the name of the sample in accordance with that indicated on the request form. In the case of RNA samples, these must be sent in a frozen state in dry-ice. For further information about sending specific samples for each analysis, please visit the Sample requirements section and  the Services offered section. The technical personnel will inform the user by email of receipt of the samples sent by Courier Service.
4. To request CGH, CNV, methylation, ChIP-on-chip, microRNAs or Exon microarray services, please contact the unit's technical personnel.
5. The Gene Expression Unit will inform the user once the experiment has been completed, and the results will be sent by email or in a CD/DVD by regular mail. The period for delivery of the results depends on the type of analysis requested and the volume of samples, and may vary between 24-48 h (2100 Bioanalyzer), 72 h- 3 weeks (real-time PCR) and 4-6 weeks (microarray service). For further information, please visit the section referring to the type of service requested.
6. In case of any change in the accepted budget, and once the results have been delivered, the Unit will then send the user a final budget specifying the type of service rendered, the price per unit, number of units, total cost of the service and the invoicing details provided by the group in the User Register form. SGIker will invoice the research group for the agreed price.
7. The samples, primers and probes sent by the user will be kept for a maximum period of 2 months following completion of the service and delivery of the results.  The technical personnel will contact the user prior to disposal of the of samples.

Recommendations for sending of samples

The samples should be sent together with the service request form, duly completed. In the case of microarray projects, it is important to attach the greatest amount of information possible about the project.
The samples should be sent in 1.5 ml tubes, properly identified by the name of the sample as indicated on the request form, and dissolved in RNase-free water or water treated with DEPC. The RNA samples should be sent in a frozen state in carbonic snow or dry ice.

Amounts required

• 2100 Bioanalyzer:

The sending of 2 ul with a concentration of 25-500 ng/ul for total RNA is required, and 25-250 ng/ul of mRNA in the case of 6000 Nano Chips RNA.

• Real-time PCR:

1 ul of cDNA per reaction is used, and the samples are migrated in triplicate. Send a sufficient quantity of sample to cover all reactions: 3 x No. of genes to be analysed. The cDNA should have a concentration of 10-100 ng/ul (use the concentration of RNA as an approximate guideline). In the case of Sybr Green, it is necessary to make a standard curve with 5 dilutions of one standard sample, preferably from the same tissue and type of extraction as the samples to be analysed, and this should be with a concentration of over 50 ng/ul.
The RNA used for synthesis of the cDNA should have a A260/280 ratio of between 1.8-2.1.
IT IS RECOMMENDED TO ANALYSE THE INTEGRITY OF TOTAL RNA WITH 2100 BIOANALYZER OR SIMILAR. RIN ≥ 7.

In the case of SYBR Green and where the amplicon has not been designed in different exones (genes from a single exon), treatment of the sample using DNase is recommended followed by sending of an RT minus sample, cDNA reaction - albeit without adding the enzymeny non-specific amplicon.

• Microarray service.

           -Gene Expression:
4x and 8x array formats: 200 ng  of total RNA with a concentration of 50 ng/ul,  dissolved in ultra-pure, RNase-free water.
2x and 1x array formats: 400 ng  of total RNA with a concentration of 100 ng/ul,  dissolved in ultra-pure, RNase-free water.
Send the samples properly identified in 1.5ml Eppendorf tubes, frozen in dry ice.
Minimum quality requirements:  A260/280 ≥ 1,8; RIN≥ 7

• miRNAs

400 ng of total RNA, with a concentration of 100 ng/ul,  dissolved in ultra-pure, RNase-free water.
Minimum quality requirements:  A260/280 ≥ 1,8; RIN≥ 7

• CGH/CNV

8x array formats: 1 ug of DNA with a concentration of 100 ng/ul, dissolved in ultra-pure water.
4x, 2x and 1x array formats: 2 ug of DNA  with a concentration of 100 ng/ul, dissolved in ultra-pure water.
All samples must be run in an agorase gel to asess DNA integrity, and a picture of the gel must be sent with the sample and service request form.
Minimum quality requirements: A260/280 ≥ 1,7; A260/230 ≥ 1,5
NOTE: In case sample requirements are not fulfilled, please consult the unit's technical personnel.

Recommended RNA and DNA purification and cDNA synthesis methods

RNA extraction methods

• Microarray analysis:

           -For RNA extraction from tissues, it is advisable to use an organic extraction followed by column purification (RiboPure kit,  AMBION).

           -For cells, organic extraction  (TRI Reagent, AMBION; Trizol Reagent, Invitrogen) or column purification (RNAqueous kit, AMBION), is sufficient.

• Real-time PCR

           -For RNA extraction from tissues, organic extraction (TRI Reagent, AMBION; Trizol Reagent, Invitrogen).

           -For cells, organic extraction  (TRI Reagent, AMBION; Trizol Reagent, Invitrogen) or column purification (RNAqueous kit, AMBION), is sufficient.

• For RNA extraction from small amounts of samples, such as those obtained from laser microdissection, two methods are recommended which are already validated for real-time PCR: the Absolutely RNA Nanoprep kit made by Stratagene; and the RNAqueous Mikro kit made by AMBION.
• For RNA extraction for analysis of microRNAs using the miRNA microarray system, Agilent Technologies recommends three extraction methods: the miRNeasy Mini kit made by QIAGEN, the mirVana RNA isolation kit made by AMBION, or Trizol Reagent (Invitrogen). Agilent recommends following the manufacturer's instructions for total RNA extraction.

DNA extraction for the aCGH microarray system made by Agilent Technologies:

• Agilent Technologies recommends the DNeasy Blood & Tissue kit made by QIAGEN.
• For genomic DNA extraction from fixed and paraffin-embedded samples (FFPE), please consult the unit's technical personnel.

Methods for cDNA synthesis from total RNA for gene expression analysis using real-time PCR.

• The use of random primers is recommended
• The Unit has successfully validated three cDNA synthesis kits: the High-capacity cDNA Reverse Transcription kit with RNAse Inhibitor, made by Applied Biosystems; the High-capacity RNA-to-cDNA Master Mix made by Applied Biosystems and the SuperScript First-Strand Synthesis System for RT-PCR, made by Invitrogen.

Quality control requirements of ARN sample

One of the critical steps for the success of the analyses carried out using real-time PCR and microarray technology is the quality of the RNA samples. The quality of the RNA is measured using three basic parameters:

• Purity: contamination of the sample by organic or inorganic impurities (e.g. phenol, chloroform), and proteins, significantly affects the sensitivity and specific nature of the result. To determine the degree of RNA purity, the A260/280 and A260/230 absorbance ratio is determined, which should be = 1.8.
• Integrity: Absorbance at 260 nm and the A260/280 ratio only gives us an indication of the amount of RNA and the degree of contamination by organic or inorganic impurities. It does not, however, tell us anything about the level of RNA degradation. There exist two methods for determining the degree of RNA degradation:

           -28S and 18S ratio between ribosomal bands which are observed in an agarose gel as discreet bands and whose 28S/18S ratio in a whole RNA is close to two. A 28S/18S = 1.2 ratio is recommended.

           -RIN - the RNA Integrity Number - is a number applied by the software algorithm of the 2100 Bioanalyzer made by Agilent Technologies. It indicates the degree of degradation of the RNA sample. An intact RNA has a RIN of 9-10, whereas a degraded RNA would have a RIN <6.

• Contamination by genomic DNA: analytical techniques for gene expression using real-time PCR or microarrays are techniques which are extremely sensitive to genomic DNA contamination of the RNA sample. DNA contamination may have a significant effect on the sensitivity and specific nature of the result of gene expression. The best method for preventing genomic DNA contamination is to use an extraction method that combines organic extraction in phenol (Trizol or TRI Reagent) and subsequent column purification. There exist column purification kits that include pre-treatment by column using DNase and subsequent elution of the RNA. In those cases in which the sample contains a degree of unacceptable contamination for the purpose of applying microarrays or real-time PCR with SYBR Green (especially important), the sample should be treated with DNase and then re-purified by column following treatment. it is very important to remove all the DNase, as this inactivates any retrotranscription reaction in the cDNA or cRNA synthesis.

           -DNA-free: a new method to remove DNA, AMBION
           -Avoiding DNA contamination in RT-PCR
           -RNase free DNase set, QIAGEN

It is essential that all the samples subject to analysis should have comparable degrees of purity (A260/280) and integrity (28S/18S and/or RIN number).
For further information regarding the quality of the RNA and how to analyse it, please read the following notes from AMBION:

• Assessing RNA quality: The Good, The Bad and the Ugly
• Is your RNA intact? Methods to check RNA integrity: AMBION TechNote 8(3)
• Assessing RNA quality: AMBION TechNote 11(1)
• Determinants of RNA integrity and Purity: AMBION TechNote 11(2)

Basic recommendations for handling RNA

RNA is an extremely sensitive material and the utmost precaution must be taken to prevent contamination by ribonucleases (RNases) and its degradation. RNases are stable and active enzymes that do not require co-factors in order to function. RNases are very difficult to inactivate, support autoclaving and are insensitive to standard methods used for the inactivation of proteins. It is important to work in an RNase-free environment, and the material must be treated in order to eliminate it. The following recommendations help to prevent contamination by RNases:

• Use gloves throughout the handling process of samples, perishable material and glass, as well as RNA extraction. RNases are present in large amounts on the skin and this is the easiest means of contamination. Try to work as fast as possible during extraction so as to minimize exposure to RNases and prevent RNA degradation.
• Optional: Treat surfaces, ends of pipettes and test tube racks with RNaseZap (AMBION) or some other RNase decontaminating solution. Use specific pipettes when handling RNA.
• Always keep the RNA sample in ice or a cold block, at least until it has dissolved in an organic solvent.
• Use disposable plastic sterile material (tubes, ends of pipettes) which has been certified free of RNases.
• Glass material must be incubated in an oven at 240ºC all night. Plastic lids and any other type of non-disposable material such as electrophoresis tubs, test tubes and precipitation vessels must be washed with 0.1M NaOH, 1mM EDTA for 5 minutes, and then rinsed in RNase-free MilliQ water. Alternatively, chloroform-resistant plastic material may be rinsed in chloroform.
• Water and solutions (except those that contain Tris-base) must be treated with 0.1 % (v/v in distilled water) DEPC* (diethyl pyrocarbonate). When preparing the 0.1 % DEPC solution. Thoroughly mix it for 1 minute, incubate at room temperature the whole night in the extractor hood, and autoclave the solution before using it to inactivate the DEPC. If the solution contains Tris-base, prepare it with autoclaved water-DEPC.
• Precaution: DEPC is toxic and possibly carcinogenic. use gloves and carry out the entire process in an extractor hood until it is inactivated by autoclaving.
• It is advisable to re-suspend the RNA in ultra-pure water treated with DEPC or RNase-free water.

Recommendations for RNA extraction

The use of fresh samples to obtain RNA is recommended. To extract RNA from tissues, there are 3 options:

• Inmediately homogenize tissue sample in lisis buffer.
• If it is not possible to proceed to immediate homogenization in lisis buffer, tissue sample must be stabilized  immediately after it has been obtained. There are two methods for tissue stabilization:

           -Freeze the tissue in liquid nitrogen immediately after it has been obtained. Tissue frozen in this way can be maintained at -80ºC until the RNA is extracted. For extraction, slice and weigh the unfrozen tissue, and keep it in dry ice until it is homogenized in lysis solution. It is recommended to isolate several small tissue samples, instead of a bigger sample.

Another solution that can be used to accumulate tissue until the time of extraction is an RNA stabilizing solution such as RNAlater made by. Follow the manufacturer's instructions. This is a very useful stabilization method to send samples at room temperature. Tissue samples must be ≤ 0.5 cm in any single dimension. Cut large tissue samples in smaller samples, not bigger than 0.5 cm in any single dimension.
For gene expression analysis, processing of the control or reference samples in parallel is essential, following exactly the same procedure.
Microarray and real-time PCR techniques are extremely sensitive techniques that require samples of great quality and integrity which are free of impurities and genomic DNA.

Access to the unit of Gene Expression involves meeting the requirements set forth in the Protocol for access to SGIker and the services it provides.