tissue: foregut, midgut, caeca, hindgut treatment: control diet, PI supplemented diet, starved developmental stage: 5th larval
Treatment protocol
Prior to dissection, 5th larval locusts were transferred to artificial diet. One group received a meal containing plant-derived PI (SBTI, SBBI: 1% of total protein content), while another group received a control diet, where BSA was added to achieve equal total protein contents in the diets. A third group of locusts were starved for three days. After three days of treatment, foreguts, midguts, gastric caeca, hindguts and brains were dissected for total RNA extraction. For each feeding condition, three pools of five animals were dissected, meaning a grand total of 45 locusts was used for the study.
Growth protocol
Gregarious locusts were reared under crowded conditions with controlled temperature (32 ± 1°C), light (14h photoperiod) and relative humidity (40-60%). Locusts were fed daily with grass.
Extracted molecule
total RNA
Extraction protocol
Total RNA from dissected gut tissues was extracted using the RNeasy Lipid tissue kit (Qiagen), while total RNA from brain was extracted using the RNeasy Mini Kit (Qiagen). DNaseI treatment was performed to remove traces of genomic DNA contamination. Quality and concentration of the extracted RNA were assessed using the Agilent 2100 Bioanalyzer.
Label
Cy5
Label protocol
For all dissected gut parts (foregut and hindgut, midgut, caeca), equal quantities of RNA were combined and used as template to produce one pool of ‘gut’ complementary RNA (cRNA). Similarly, RNA from all brain samples was combined into a single pool of brain RNA for subsequent generation of cRNA. Labeling of the samples was performed by using the Quick Amp Labeling Kit (Two Color, Agilent Technologies), according to the manufacturer’s instructions. For each sample, 1µg total RNA was used for labeling. The fluorescently labeled samples were purified by means of the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions.
Channel 2
Source name
Foregut/midgut/caeca/hindgut, control/PI/starved diet, pooled sample
tissue: foregut, midgut, caeca, hindgut treatment: control diet, PI supplemented diet, starved developmental stage: 5th larval
Treatment protocol
Prior to dissection, 5th larval locusts were transferred to artificial diet. One group received a meal containing plant-derived PI (SBTI, SBBI: 1% of total protein content), while another group received a control diet, where BSA was added to achieve equal total protein contents in the diets. A third group of locusts were starved for three days. After three days of treatment, foreguts, midguts, gastric caeca, hindguts and brains were dissected for total RNA extraction. For each feeding condition, three pools of five animals were dissected, meaning a grand total of 45 locusts was used for the study.
Growth protocol
Gregarious locusts were reared under crowded conditions with controlled temperature (32 ± 1°C), light (14h photoperiod) and relative humidity (40-60%). Locusts were fed daily with grass.
Extracted molecule
total RNA
Extraction protocol
Total RNA from dissected gut tissues was extracted using the RNeasy Lipid tissue kit (Qiagen), while total RNA from brain was extracted using the RNeasy Mini Kit (Qiagen). DNaseI treatment was performed to remove traces of genomic DNA contamination. Quality and concentration of the extracted RNA were assessed using the Agilent 2100 Bioanalyzer.
Label
Cy3
Label protocol
For all dissected gut parts (foregut and hindgut, midgut, caeca), equal quantities of RNA were combined and used as template to produce one pool of ‘gut’ complementary RNA (cRNA). Similarly, RNA from all brain samples was combined into a single pool of brain RNA for subsequent generation of cRNA. Labeling of the samples was performed by using the Quick Amp Labeling Kit (Two Color, Agilent Technologies), according to the manufacturer’s instructions. For each sample, 1µg total RNA was used for labeling. The fluorescently labeled samples were purified by means of the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions.
Hybridization protocol
The Cy5- and Cy3-labeled samples were co-hybridized for each tissue onto the microarrays by using the Gene Expression Hybridization Kit (Agilent Technologies) following the manufacturer's instructions. Hybridization was performed at 65°C for 17h. Next, the slide was washed: twice in GE Wash Buffer 1 (Agilent) for 1min each, in GE Wash Buffer 2 (Agilent) at 37°C for 1min and in acetonitrile (Sigma) for 1min. Finally, the slides were immersed in Stabilization and Drying Solution (Agilent) for 30sec.
Scan protocol
The microarray slide was scanned using a GenePix Personal 4100A confocal scanner (Axon Instruments) at a resolution of 5 µm and excitation wavelengths of 635 nm and 532 nm. The photomultiplier tube voltages for separate wavelengths were adjusted to obtain an overall green/red ratio as close as possible to 1. Theoretically, ratios for all probes are expected to be 1, since we are dealing with self-self hybridizations from a cRNA sample that is labeled in both red (Cy5) and green (Cy3). Spot identification and quantification of the fluorescent signal were carried out using GenePix Pro 6.0 software (Axon Instruments).
Description
Gut
Data processing
For each transcript, two different probes were spotted on the array. Only one probe per expressed transcript was retained for analysis and future use. For transcripts where both probes emitted a signal, but one probe produced a lower signal for both red and green, the weakest probe was omitted. When neither one of the probes had the lowest intensity for both the red and the green signal, the probe with the smallest deviation from green/red ratio = 1 was selected. Transcripts for which the median foreground (FG) intensity was lower than the average local background (BG) intensity (calculated over the complete array) plus one standard deviation (SD) for both the brain and gut array were considered unexpressed and were deleted before analysis. Any spot with an intensity of FG > BG + 1SD in either tissue was initially considered to be expressed in that tissue. To be able to roughly compare spot intensities between brain and gut, transcript intensity was calibrated against spiked-in controls, which were added in the same quantity in both samples. This was done by dividing the average signal intensity of the spiked-in control group spots from the brain sample by that of the gut sample and multiplying the original signal intensity of the gut transcripts with the obtained factor. The transcripts were sorted based on the average Cy5 and Cy3 intensity in both brain and gut. In addition to transcripts that had a FG > BG + 1SD in only one of both tissues, transcripts with an average intensity ratio (when comparing the two tissues) of > 20 were also considered tissue specific.
