The overall goal of the following experiment is to study transcriptional dynamics and defined brain nuclei of mice following behavioral experience. This is achieved by first habituating the animals and recording their locomotor activity after saline injection. Once habituated, locomotor activity is recorded after a single dose of cocaine, samples from the nucleus accumbens are then collected and used for RNA isolation and CDNA preparation.
Next QPCR analysis is performed in order to study gene expression induced by behavior. Ultimately, results are obtained that show transcriptional dynamics following cocaine experience Demonstrating the protocol will be Dr.Hatu, the lab manager, Dum Muer, a graduate student from the lab and Duran an undergraduate student from the lab For three consecutive days. Each mouse is habituated to the handling and injection procedures, organized mice into cages prior to initiating the experiment, such that mice will not be transferred between cages in the future and mice will never be left in isolation for an extended period of time.
30 minutes before the start of testing, all test cages should be transferred into the behavioral room to allow the animals to acclimate to the environment. Administer an intraperitoneal injection of 250 microliters saline immediately following the injection, place the mouse in a behavior chamber to monitor locomotor activity for 15 minutes. The mouse could be placed in the behavior chamber for 20 minutes.
An activity monitored from minutes two to 17. Repeat these steps at the same time of the day for the following two days. On the fourth day, administer a single injection of cocaine or saline, depending on the group, and immediately place the mouse in the chamber to monitor local motor activity for 20 minutes at selected time points following cocaine exposure, extract the brain and place it in ice cold, A CSF solution for one to two minutes to chill and harden.
After removing the cerebellum and wicking away excess fluid, glue the brain to the stage of a vibram with the rostral part facing up. Fill the chamber with ice cold A CSF and slice 200 micron sections until the nucleus accumbens is clearly identified. Pay close attention to the shape of the corpus callosum, the location and shape of the anterior commissure and the ventricles while cutting.
Once the correct region is identified, slice two sections of 400 microns from which the nucleus accumbens will be dissected under a stereoscope. Use a fine blade to dissect the nucleus accumbens away from the slices. Four quick passes of the blade in the correct area should isolate the appropriate tissue immediately.
Place the isolated tissue into 900 microliters of lysis reagent, and store at minus 80 degrees Celsius until RNA extraction to begin thaw the tubes at 37 degrees Celsius and homogenize. Using a 25 gauge needle, the first few rounds may be difficult and require pushing the tissue against the wall of the tube to homogenize it into small pieces. To ensure proper homogenization, pipette the lysate onto a kaya shredder mini spin column and centrifuge.
Pipette the lysate into a new micro centrifuge tube and extract the RNA according to the manufacturer's instructions. Following RNA purification reverse transcription to CDNA is performed as well as target specific preem amplification of the samples with primers directed against the group of genes chosen for analysis. These procedures are described in the text version of the protocol.
Commercial microfluidic chips for comprehensive QPCR analysis are available in 48 by 48. Format, enabling analysis of 48 samples with 48 probe sets, or in a 96 by 96 format in which 96 samples are analyzed with 96 probes. In most cases, the choice of chip primarily depends on the number of samples analyzed.
First, prepare the sample mix according to the chip chosen for the experiment. Use caution when pipetting the 20 XDNA binding dye sample loading reagent. Agitate the sample mix solutions for a minimum of 20 seconds and centrifuge for at least 30 seconds.
Prepared reactions can be stored for short durations at four degrees Celsius until the chip is ready for loading. Next, prepare the assay mix, agitate the sample mix thoroughly and centrifuge to spin down all of the components. As shown earlier, the dynamic array IFC chip has inlets for samples and assays, as well as specified inlets for control line fluid used to pressurize the microfluidic chambers.
First, inject control line fluid into each accumulator on the chip. Next, prime the chip by placing it into the IFC controller and running the appropriate script. When the script has finished, remove the primed chip from the IFC controller and pipette five microliters of each assay.
Mix onto the chip. Add five microliters of each sample mix into their inlets and return the chip to the IFC controller using the IFC controller software. Run the script to load the samples and assays into the chip.
When the load mix script has finished, remove the loaded chip from the IFC controller. Next, load the chip on the thermal cycler and create a new chip run that includes a melting curve analysis. Using the thermal cycler software ensure that the reaction chambers are recognized properly and allow the reaction to run to completion.
An increase in locomotor activity was apparent after a single acute cocaine injection. In this coronal section, thick white lines marked the location of cuts made to define the boundaries of the nucleus accumbens. During dissection, the amplification curves of primers directed against CFOs and phos B demonstrate that the primer pairs amplify their target transcript with approximately 100%efficiency.
In every cycle, results show that cocaine induced expression of CFOs and phos B in the nucleus accumbens was highest one and two hours after exposure. Following this procedure, other methods like next generation sequencing can be performed in order to identify novel uncharacterized transcript.
