Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

This microarray hybridization method is useful for comparing a large number of samples from an organism with a known genome. Compared to a high throughput sequencing approach, the amount of data generated by this method is much easier to handle and further, it can be analyzed in a more cost effective way. Demonstrating the procedure will be Dr.Christiane Seiler a post-doctoral scientist working in the research group at Rosis at the IPK in Gatersleben.

Before performing microarray hybridization using the gene expression hybridization kit, prepare 10X blocking agent according to manufacturer's specifications and aliquot the resulting blocking agent in 200 microliter volumes. Then, store the blocking agent at minus 20 degrees Celsius until use. On the day of the microarray hybridization, thaw one 200 microliter aliquot of the 10X blocking agent on ice and prewarm the hybridization oven to 65 degrees Celsius and a heat block to 60 degrees Celsius.

Prepare the fragmentation mix for each sample as described by the manufacturer and mix the samples gently on a vortex. After vortexing, briefly spin down the samples in a microcentrifuge before incubating for exactly 30 minutes in the 60 degree Celsius heat block. At the end of the incubation, immediately cool each tube on ice for one minute before stopping the fragmentation with 25 microliters of 2X gene expression hybridization buffer, high revolutions per minute per tube.

Mix with gentle pipetting, taking great care to not introduce any bubbles, and centrifuge the tubes in ambient room temperature. At the end of the spin immediately place all of the tubes on ice and load each sample into the microarray slide as quickly as possible. Next, slowly dispense 44 microliters of each hybridization mix into the center of each gasket well, taking care to avoid introducing bubbles, and add 44 microliters of hybridization buffer into any unused wells.

Immediately place the microarray slide in the correct orientation on top of the gasket slide, taking care not to spill any liquid, and tightly close the hybridization assembly. Rotate the assembly to confirm the lack of stationary air bubbles and place the hybridization chamber assembly into the hybridization oven rotator. Set the rotation speed to 10 revolutions per minute, then start the hybridization at 65 degrees Celsius for exactly 17 hours.

While the samples are hybridizing, prepare three wash dish assemblies in the appropriate wash buffers as outlined in the tables. After exactly 17 hours of hybridization, disassemble one hybridization chamber on a lab bench lined with lint free paper and transfer one microarray sandwich to a dish containing wash buffer one with the microarray barcode facing up in a slanted position without submerging the entire slide in the buffer. Using forceps, separate the two glass slides and let the gasket slide drop gently into the bottom of the dish while keeping a firm grip on the microarray slide.

Slowly lift the microarray slide sideways for immediate transfer into the microarray rack in dish two with a minimal exposure to the air. When all eight slides have been evenly placed along the rack, attach the rack holder and transfer the entire dish two setup to the magnetic stirrer. Stir the setup gently for exactly one minute.

While the samples are being stirred, transfer dish three from the 37 degree Celsius incubator onto a second magnetic stirrer. Then gently add 37 degree Celsius wash buffer two to dish three without forming bubbles. At the end of the stirring incubation, gently and slowly transfer the slide rack to dish three and remove the rack holder.

After stirring for exactly one minute, slowly and gently remove the slide rack from the dish and use lint free paper to carefully dry both sides of each slide, placing each slide into a slide box as it is dried. After allowing the slides to dry for 15 minutes, transfer each microarray slide into a slide holder with the barcode facing up and load the assembled slide holders into a scanning carousel and sequence according to the barcode number. Then, immediately scan the slides.

In this figure, a representative result from a run to test the quality of RNA extraction is shown. In typical analyses of low starch content samples, such as barley leaves, additional ribosomal RNA bands from chloroplasts are evident. High starch content samples, such as barley seeds, exhibit 18 and 28S ribosomal RNA.

Note that no automated RNA integrity number value can be calculated for green plant tissues, such as leaf samples, due to chloroplast ribosomal RNA. In this representative analysis, an RNA preparation and hybridization to a customized barley microarray chip were performed as demonstrated. The grid shows an example of derived signals from each corner of the chip, including background and spike in readout dots used for calibration.

The histogram indicates the deviation of detectable dots with respective signal intensities. As observed, a successful hybridization gives a broad Gaussian-shaped curve with only minor outliers. Shown here is the quality control report for detected signals.

The values for the hybridized slide are given in column two and the range of acceptable values is shown in column four. This method can be used for addressing any experimental set up for any organism where substantial information about the genome is available. Using this technique in our lab enabled us to compare and analyze transcriptomic changes in rice and barley that occur after growth stress induction.