Update 2 files

- /assays/qRT-PCR/protocols/qRT-PCR_protocol.txt
- /assays/qRT-PCR/protocols/qRT-PCR_protocol.md

assays/qRT-PCR/protocols/qRT-PCR_protocol.md

+## qRT-PCR protocol
+
+**Sample mix:**
+
+	4 µl	cDNA
+	5 µl	2X Luna Universal qPCR Master Mix (NEB)
+	0.02 mM	fwd/rev primer
+	0.75 µl	H2O
+
+- two technical replicate for each biological sample
+
+Standard curve:
+
+- instead of sample cDNA: use purified plasmid that carries the amplified sequence of the respective target gene in known concentration
+- six 1:10 dilutions, starting with 40 pg
+
+**qPCR run:**
+
+LightCycler 480 (Roche), 3-step PCR:
+
+		95°C	5 min
+
+		45 cycles:
+		95°C	10 sec
+		60°C	10 sec
+		72°C 	10 sec
+
+		Melting curve:
+		95°C 	5 sec
+		65-97°C	1 min each
+
+**qPCR analysis: calculate relative gene expression from the raw data:**
+
+**1. Quality control**
+
+- check quality of the run in the LightCycler480 software (Roche; version 1.5.1.62)
+- remove samples with unspecific melting peaks
+- remove samples with a Cp value >40 (and consider them as 0 expression)
+
+**2. Extract data**
+
+- if internal standard concentration was available (plasmid): use LightCycler480 software to calculate concentrations for each samples
+- export Cp values (and concentrations) to txt file
+
+**3. Calculate relative expression for all biological replicates**
+
+If a standard curve was available:
+- calculate mean concentration values of technical replicates
+- calculate relative expression for each biological replicate by dividing the mean expression (of technical replicates) of the target gene by the mean expression (of technical replicates) of the housekeeping gene Actin
+
+If no standard curve was available:
+- calculate mean Cp values of technical replicates
+- calculate relative expression for each biological replicate by delta Ct: 2^( (Actin(mean Cp of technical replicates)) - ((Target gene (mean Cp of technical replicates))
+
+**4. Calculate means and standard deviation for all samples**
+
+- relative expression was averaged over the three biological replicates for each sample
+- standard deviation was calculated across the three biological replicates for each sample
\ No newline at end of file

assays/qRT-PCR/protocols/qRT-PCR_protocol.txt

-sample mix:
-
-	4 µl	cDNA
-	5 µl	2X Luna Universal qPCR Master Mix (NEB)
-	0.02 mM	fwd/rev primer
-	0.75 µl	H2O
-
-	- two technical replicate for each biological sample
-
-standard curve:
-	- instead of sample cDNA: use purified plasmid that carries the amplified sequence of the respective target gene in known concentration
-	- six 1:10 dilutions, starting with 40 pg
-
-qPCR run:
-
-	LightCycler 480 (Roche), 3-step PCR:
-
-		95°C	5 min
-
-		45 cycles:
-		95°C	10 sec
-		60°C	10 sec
-		72°C 	10 sec
-
-		Melting curve:
-		95°C 	5 sec
-		65-97°C	1 min each
-
-qPCR analysis: calculate relative gene expression from the raw data
-
-	1. quality control
-
-	- check quality of the run in the LightCycler480 software (Roche; version 1.5.1.62)
-	- remove samples with unspecific melting peaks
-	- remove samples with a Cp value >40 (and consider them as 0 expression)
-
-	2. extract data
-
-	- if internal standard concentration was available (plasmid): use LightCycler480 software to calculate concentrations for each samples
-	- export Cp values (and concentrations) to txt file
-
-	3. calculate relative expression for all biological replicates
-
-	if a standard curve was available:
-	- calculate mean concentration values of technical replicates
-	- calculate relative expression for each biological replicate by dividing the mean expression (of technical replicates) of the target gene by the mean expression (of technical replicates) of the housekeeping gene Actin
-
-	if no standard curve was available:
-	- calculate mean Cp values of technical replicates
-	- calculate relative expression for each biological replicate by delta Ct: 2^( (Actin(mean Cp of technical replicates)) - ((Target gene (mean Cp of technical replicates))
-
-	4. calculate means and standard deviation for all samples
-	- relative expression was averaged over the three biological replicates for each sample
-	- standard deviation was calculated across the three biological replicates for each sample
\ No newline at end of file