An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing

Table 3 Comparison of Cq values obtained using RNA isolated with the old vs. the new protocol

Primer	Sample	Mean Cq old	Mean Cq new	Δ Mean Cq new – mean Cq old
GAPDH	#2 Normal	26.99	26.56	−0.43
	#2 Tumour	24.89	24.61	−0.29
	#3 Tumour	22.24	17.75	−4.49
	#4 Tumour	22.99	19.23	−3.75
	#5 Normal	26.74	24.40	−2.34
	#6 Tumour	20.97	20.98	0.00
	#7 Tumour	23.57	19.79	−3.78
	#8 Normal	23.72	23.28	−0.44
	#9 Normal	27.36	27.77	0.41
	#9 Tumour	24.82	19.33	−5.49
	#10 Normal	27.29	26.89	−0.40
	#10 Tumour	N.d.	25.68	n.a.
	Mean ΔCq New−Cq Old for GAPDH			−2.06
B2M	#2 Normal	25.83	24.62	−1.21
	#2 Tumour	21.88	21.27	−0.61
	#3 Tumour	22.76	17.48	−5.28
	#4 Tumour	22.70	18.83	−3.87
	#5 Normal	24.33	21.38	−2.95
	#6 Tumour	21.32	21.07	−0.25
	#7 Tumour	23.89	18.87	−5.02
	#8 Normal	24.13	24.46	0.33
	#9 Normal	27.63	26.14	−1.49
	#9 Tumour	25.94	21.36	−4.58
	#10 Normal	28.98	26.73	−2.25
	#10 Tumour	27.92	24.16	−3.76
	Mean ΔCq New−ΔCq Old for B2M			−2.59

Identical amounts of RNA were reverse transcribed and analysed by RT-qPCR using GAPDH or B2 M primers. First column: primers that were used for the analysis. Second column indicates the case and type of tissue from which the RNA that was analysed derived. “Mean Cq Old” denotes the mean Cq values obtained with RNA isolated using the old extraction protocol. “Mean Cq New” shows the Cq values obtained with RNA isolated using the new extraction protocol. “Δ Mean Cq New-mean Cq old” shows the difference in Cq values between the new extraction protocol and the old extraction protocol; a negative value here indicates better performance (lower Cq values) of the new extraction protocol. “Mean Δ Cq New-Cq old values” for GAPDH and B2 M list the mean difference of the Cq values between new and old extraction across all samples. N.d not detectable, n.a not applicable.

ISSN: 1471-2199