Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

Table 1 Different protocols tested to optimize tissue section preparation before laser capture microdissection of mammary epithelial, yield and integrity of RNA extracted from microdissected cells.

	N°1	N°2	N°3	N°4	N°5	N°6	N°7	N°8
Ethanol 95%						30 s
Ethanol 75%	30 s	30 s	30 s	30 s	30 s	30 s	60 s	30 s
Ethanol 50%						20 s	20 s
RNA protector			RNA later^® 100 μl, 30 s	RCL2^® 100 μl, 30 s	RCL2^® 100 μl 30 s
Water	+		30 s	30 s				+
Staining	HistoGene^® 15 s	HistoGene^® 10 s	HistoGene^® 10 s	HistoGene^® 10 s	Cresyl Violet^® 20 s	Cresyl Violet^® 20 s	Cresyl Violet ^® 20 s	-
Ethanol 50%						20 s	5 s
Water	+							+
Ethanol 75%	30 s	5 s	5 s	5 s	5 s	30 s	30 s	30 s
Ethanol 95%	2 × 1 m	2 × 1 m	2 × 1 m	2 × 1 m	2 × 1 m	30 - 40 s	2 × 1 m	2 × 1 m
Ethanol 100%	2 × 1 m	2 × 1 m	2 × 1 m	2 × 1 m	2 × 1 m	30 - 40 s	2 × 1 m	2 × 1 m
Xylene	2 × 5-10 m	2 × 5-10 m	2 × 5-10 m	2 × 5-10 m	2 × 5-10 m	2 × 5-10 m	2 × 5-10 m	2 × 5-10 m
Time of LCM	30-40 m	30-40 m	30-40 m	40-50 m	40-50 m	40-90 m	40-90 m	30 m
RNA integrity (ΔRIN)	-3.5 to -6	-3 to -4	- 2	-2 to -3	-0.5 to -1	-0.5 to 1	- 0.5 to -1	- 2
Morphology	++++	+++	----	+++	+++	++	+++

In protocols 1 to 4 tissue sections were stained using the HistoGene^® LCM frozen section Staining Kit (Arcturus). Cresyl Violet^® staining (protocols 5 to 7) using Ambion LCM staining Kit provides good morphology and yields high quality RNA. The best protocol was determined to be No.7 (framed and bold), because of the high RNA integrity and morphology and because it was easier to handle without any RNA protector and quicker to perform.

ISSN: 2661-8850