定量的リアルタイムPCRによるマウス乳腺腫瘍モデルにおける肺転移の評価

The overall goal of this procedure is to detect the quantity of a specific MRNA from tumor cells in the lung, as a method for analyzing metastasis. The main advantage of this technique is that it is a quantitative measure of metastasis. To perform a lung tumor dissection, after euthanising a mouse using a lethal dose of isofluorine according to the text protocol, place the mouse on a foam board on its back with the limbs spread, and use dissection pins to secure it to the board.

After using ethanol to spray the mouse, use forceps to grasp the lower portion of the animal at the center, and with scissors, cut upwards towards the neck through the skin, being careful not to pierce the peritoneal cavity of the mouse. Now, grasp the peritoneum at the lower end of the animal, and begin cutting upwards toward the base of the neck. Carefully cut along the left and right sides of the ribcage, taking care not to sever any blood vessels that may bleed into the thoracic cavity.

Remove the rib bones by cutting to the left and right, through the diaphragm and upper portions of the ribcage. Then, using forceps, grasp the trachea of the mouse and pull forward, and use dissection scissors to sever the trachea before removing the lungs from the mouse. Use PBS to gently wash the lung tissue.

Then, after gross evaluation of the lung tissue according to the text protocol, if the lungs are to be analyzed immediately, proceed to RNA isolation. To isolate RNA from tissue, begin by homogenizing the tissue using one of the methods listed in the text protocol. Prepare lysis buffer from an RNA isolation kit, and add 20 microliters of tumor captoethanol to each milliliter of lysis buffer.

Resuspend every 30 grams of tissue in 300 microliters of lysis buffer, and with a sonicator set at 30%amplitude, sonicate the tissue for 10 seconds. Add 10 microliters of protinase K to 590 microliters of TE buffer, and add 600 microliters of the buffer to 300 microliters of sonicated tissue. Incubate at room temperature for 10 minutes.

Next, centrifuge the samples at greater than or equal to 13, 000 times G for 10 minutes to remove the debris. Then transfer the supernatant to new tubes. Add 450 microliters of ethanol to every 900 microliters of supernatant to precipitate the RNA.

Then transfer 700 microliters of the lysate ethanol mix to an RNA column. Spin again at greater than or equal to 13, 000 times G for one minute to bind the RNA to the column. Then discard the liquid waste, and add the remaining supernatant to the column, and spin again.

Next, add 350 microliters of wash buffer one to the upper portion of the column, and centrifuge the samples for 30 seconds. Discard the liquid waste and replace the column in the tube. Prepare DNase by mixing five units of DNase one enzyme with five microliters of 10X DNase buffer, and 40 microliters of DNase RNase free water for each sample.

Add 50 microliters of DNase mix to each column, and incubate at room temperature for 15 minutes. Then add 350 microliters of wash buffer one to the columns and centrifuge the samples for 30 seconds. After discarding the waste solution, add 600 microliters of wash buffer two, and spin for an additional 30 seconds.

Discard the liquid before adding 250 microliters of wash buffer two, and then spin for two minutes. Transfer the column into a new collection tube, and add 50 to 100 microliters of RNase DNase free water to the column. After incubating for one minute, spin for one minute.

Re-run the collected eluate through the column to increase the RNA yield. For immediate use, keep the samples on ice. For later use, snap freeze on dry ice or liquid nitrogen, and store at minus 80 degrees Celsius until needed.

Using a spectrophotometer, quantify the RNA. Then, in sterile RNase DNase free PCR tubes or plates, use 0.5 to 2 micrograms of total RNA to prepare the reaction mix for first transynthesis, as shown in this table. Mix, and gently centrifuge, and program a standard PCR machine using the program shown here.

When the reaction is complete, use sterile, nuclease free water to dilute the finished reaction to the desired volume. To carry out real time PCR, use a positive control C DNA, such as human HER2, to set up a standard curve for each primer set by preparing one to four serial dilutions of the C DNA to generate five standards. Next, calculate the number of total samples, which should include the standard curve and experimental samples.

Then, in sterile RNase DNase free tubes, prepare a master mix for each experimental gene of interest, as well as an internal control, such as gapdh. Prepare a test plate containing one microliter of C DNase for each standard or experimental sample. Allocate at least one C DNA sample per well, for each primer probe.

Add 19 microliters of mastermix per well, for each primer probe. This bioluminescence experiment demonstrates that mouse lungs treated with ACT1, which targets the gap junction protein Connexin 43, appear negative for luciferase activity, suggesting the absence of tumor cells in the tissue. However, analysis of H&E sections of the lung tissue shows micrometastases that were not illuminated by luciferase, suggesting luciferase imaging is not sensitive enough to detect low numbers of tumor cells.

Shown here are the results from a qRT-PCR experiment, using a probe specific for HER2, to detect human HER2 plus JIMT1 tumor cells in lung tissue from cells originally transplanted into the mouse memory fat pad. Although no grossly visible metastases were observed on any of the lungs, in two of the 11 samples, qRT-PCR detected metastases. In this figure, a standard curve analysis was prepared to determine the relative HER2 levels normalized to mouse lung gapdh in a cell number series.

The results indicate the qRT-PCR, using the HER2 probe, was sensitive enough to detect HER2 levels in as few as 10 cells. Finally, this regression analysis shows the relative cell number in each lung sample, including the positive and negative controls. Once mastered, this technique can be done in six to eight hours, if performed properly.

While attempting this procedure, it's important to remember to use precaution to maintain RNA integrity during RNA isolation and reverse transcriptase reaction. After watching this video, you should have a good understanding of how to detect metastasis in whole lung tissue, by quantitative real time PCR.