"Transcriptomics & Functional Genomics"

Welcome to Transcriptomics & Functional Genomics News Letter No 5. This editions focus is on RNA Quantification and Qualtiy.

Currently only the focus topic from each newsletter is being made available on the internet (please note this material is covered by copy right and permission should be sought to reproduce any content). The full newsletter is available internally via the intranet as a pdf. If you are interested in advertising a seminar or promotion via the newsletter or sponsorship please contact : Dr K Laing (Senior Scientist  Intracellullar Pathogen Cooperative).

The measurement of gene expression using QRT-PCR and microarray analysis are techniques which ultimately assess the steady state level of mRNA and as such is heavily reliant upon the integrity and quality of the RNA. This edition aims at focusing on some of the techniques and solutions that address these issues.

I hope as always you find this focus article useful and informative.

If you are interested in other topics cover in other newsletters go to:

http://www.sgul.ac.uk/depts/medmicro/newsletter.htm

Ken Laing Intracellular Pathogen Co-operative, Cellular & Molecular Medicine, St George’s University of London

RNA Integrity

Fig 1. Total RNA with an rRNA(18/28s) ratio of 2.0 and an RNA integrity number (RIN) of 9.8

By now most readers of will of heard of and many users of the BIOMICs facility may have used the instrument for RNA quality assessment, assessing your RNA quality is the first step in getting good data. The Agilent Bioanalysers main use is to perform native capillary electrophoresis of a denatured RNA sample. Other applications are available to separate and size DNA molecules, proteins and simple cell flow cytometry but will not be discussed. The instrument works by priming a “chip” with a gel dye matrix and in the case of RNA loading up to twelve heat denatured samples and an RNA ladder. Each sample well contains two calibration markers used in combination with the ladder to determine the size and quantity of RNA loaded. The instrument sequentially runs the ladder and samples quantitating the fluorescence emitted by a red fluorescent intercalating dye as the RNA passes a fixed point within the capillary. One of the major benefits of this system over conventional agarose gel electrophoresis is the sensitivity and related to this the quantity of RNA required for analysis. The standard RNA 6000 nano-kit has a qualitative range for total RNA of between 5 and 500ng/ul and a quantitative range of between 25 and 500ng/ul, whilst the RNA 6000 pico-kit has a quantitative range of between 200 and 5000pg/ul. The loading sample volume in each case is 1 ul

Fig 2. Reconstructed gel image of a RNA 6000 nano chip run

The visual output from the instrument is in two formats an RNA time/size -intensity line graph (see fig 1) or a reconstructed digitised gel image (fig 2) allowing a qualitative assessment of RNA. Typically total RNA has a ribosomal RNA ratio of around 2, a characteristic due to the relative size of the two mature rRNA species and their relative abundance. The observation of 5s/tRNA peaks is also often dependant upon the method of purification particularly where size exclusion or some columns are used. Messenger RNA or amplified RNA using for example the Eberwine technique, each has its own distinctive profile.Degradation of RNA leads to a characteristic shift in the ribosomal ratio towards 1.2-1.5 before complete loss of any well-defined rRNA peaks (see fig 3) and the accumulation of a broad fast peak with a modal size similar to tRNA.

The Expert Software 2100 was up dated last year (2004/2005), the release version integrated the instrument operation and analysis into a single package which is freely available for installation on any PC with a windows operating system and can be used to review results previously run. The use of the ribosomal RNA ratio although useful as a measure of RNA “quality” is not without its problems. The calculation of a ratio necessitates the integration of the area under the ribosomal peaks and is a process sensitive to the way in which the

Fig 3 Left Highly degraded Total RNA with rRNA ratio of 0 and RNA integrity number (RIN) of 2.9: Middle Total RNA with a rRNA (18/28s) ratio of 0.2 and an RNA integrity number (RIN) of 5.0 Right Total RNA with a rRNA (18/28s) ratio of 1.4 and an RNA integrity number (RIN) of 7.2

baseline and therefore the peak are defined. This problem especially relates to the 28s ribosomal RNA, its peak tends to be broader and the inter-ribosomal space often has a higher baseline. In an attempt to resolve this Agilent introduced a Beta version of the software incorporating the RNA integrity Number algorithm. This version is only available upon request to Agilent and the standard release does not incorporate RIN. The RIN algorithm promises to be the next automated standard in assessing RNA quality for Eukaryotes. QC using this feature is reliant upon the observation that RNA degradation is a process in which progressive shortening of the RNA occurs. When seen in the context of a mixed population of total RNA leads to a skewed appearance or low shoulder of the 28s ribosomal peak but also to a lesser extent the of 18s peak and a raised baseline in the inter-ribosomal region. As degradation progresses the baseline of the pre-ribosomal region becomes “spiky” and is raised with the accumulation of fast migrating species around the 5s/tRNA region. The algorithm divides the RNA profile into nine different regions and applies a statistic to these giving a continuous value from 10 down to 1 defining the extend of RNA degradation, 10 being the highest quality. The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Using intact RNA is a key element for successful microarray or RT-PCR analyses. The Agilent 2100 bioanalyser and RNA LabChip® kits play an important role in assisting researchers in the determination of RNA quality. See Agilent article on RNA Integrity No (RIN).

Sample Quantification and Purity Whilst the Bioanalyser provides excellent QC on RNA integrity and can be used to

Fig. 4 RNA samples extracted using Qiagen’s RNeasyTMKit eluted in TE and QC’d using the NanoDropTM scanning spectrophotometer blanked against H2O.

quantitate RNA concentration it can present problems due to the need to calculate this via interpolation of the RNA ladder and relies upon the accurate loading of and integrity of both ladder and sample and in the best case is reported to be accurate to within 10%. In addition the bioanalyser as a gel system is sensitive to the contaminating proteins, salts, detergents or phenolics and is not designed to detect these contaminants especially at low levels where the gel is unaffected but subsequent enzymatic reactions may fail as a consequence. In these situations quantity and purity is best assessed by more conventional means such as scanning spectrophotometry. The down side of this technique is the relatively large sample volumes are required. A development in this area was the introduction of the NanoDropTM cuvette free spectrophotometer (NanoDrop Technologies, although in 2007 several other instruments were introduced to compete with the Nanodrop for market share) that uses 1-2ul sample volumes, as such minimises sample losses and allows easy monitoring of samples through a series of processing steps assessing both yield and purity. Accurate quantitation and quality estimates of RNA used, whether a labelling reaction or cDNA synthesis, has real benefits in assessing and validating the results. It aids interpretation and allows the building-in of comparability to experimental design; it contributes to a decisive outcome of experiments through the exclusion of bad data due to poor quality samples.

The NanoDropTM works on a similar principle to most conventional spectrophotometers however the cuvette is replaced by quartz pedestal upon which the sample is place and forms a 1mm column of liquid between it and the sample arm. The instrument has a nominal linear range of 2-3000ng/ul of RNA or 2-3700ng dsDNA, it is reported to be accurate to within 2% and covers a spectral range of 220-750nm. Conventionally the concentration RNA has been measured at a fixed or dual wavelength of 260nm or 260nm and 280nm. This approach is useful but can be misleading where contaminants are present such as Trizol or phenol that have overlapping spectra (fig 5). Qiagen’s RNeasy lysis buffer (RLT) has a weak absorption maximum of about 235nm but does not absorb at 260nm and would only be expected to contaminate RNA if inadequate washing of the column had been performed. Although for obtaining RNA quantity and quality the NanoDrop is extremely valuable it can be used for many of the down stream processes such a monitoring dye incorporation into cDNA or proteins for gene expression profiling or CGH by microarray or 2D-gels and has a detection limit of 0.12pmol/ul for Cy5 and 0.2pmol/ul for Cy3 in the case of cDNA labelling but may also be used in a wide range of other spectrophotmetric applications.

Fig 5 Left -Trizol is a common reagent used in the extraction of RNA from various cells and tissues but has as a contaminant of has a strong absorption peak at 260-270nm coincident with that of nucleic acids, however it also has a similar absorption maxima at 230nm and contamination of samples with Trizol has a characteristic dual peak absorption. Identification of samples with low 260/230 ratios, Right- Similarly Phenol (light green) and Phenol/Chloroform (dark green) has absorption maxima at 270nm but a much low absorption at 230nm and a right shift or shoulder at 270nm with a lower. Minute amounts of Trizol or Phenol will adversely affect any enzymatic reaction

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