Using the NEBNext Poly(A) Magnetic Isolation Module to isolate mRNA prior in-house library prep

Protocols used

• NEBNext Poly(A) Magnetic Isolation Module
• TapeStation protocol High Sensitivity RNA
• High Sensitivity RNA QBIT protocol

Using the NEBNext Poly(A) Magnetic Isolation Module to isolate intact poly(A)+RNA from previously isolated total RNA. The idea is to use this kit before performing the library prep with the Zymo SwitchFree kit, and try to reduce the amount of rRNA present in the libraries.

The RNA samples I’m using here are from Montipora capitata spat (posts here, here and here) and from Pocillopora acuta adults (Zoe’s samples)

I followed the kit protocol linked above (express protocol, pages 1-3) and used 30ng of input RNA. The kit is designed to isolate intact poly(A)+ RNA from 1-5µg of previously isolated total RNA.

All samples were eluted in 15uL of elution buffer (NEBNext Tris Buffer).

Sample prep

Here’s a breakdown of input RNA volume and quantity (the kit requires a total starting volume of 50uL):

sample_id		RNA_QBIT_AVG (ng/uL)		RNA (uL)		Ultrapure water (uL)		Total input RNA (ng)
B8		21.2		1.42		48.58		30
C10		23.7		1.27		48.73		30
C9		48.9		0.61		49.39		30
POC_R12_C3		28.6		1.05		48.95		30
POC_R1_H1		24.6		1.22		48.78		30
POC_R3_C3		17.7		1.69		48.31		30

Qubit Results

I used High Sensitivity RNA Qubit Protocol linked above. RNA samples were read twice, standard only read once.

• Standard 1: 48.04
• Standard 2: 939.78

QBIT date		sample_id		Species		RNA_read1		RNA_read2
20250925		B8		Montipora capitata		too low (out of range)		too low
20250925		C10		Montipora capitata		too low		too low
20250925		C9		Montipora capitata		too low		too low
20250925		POC_R12_C3		Pocillopora acuta		too low		too low
20250925		POC_R1_H1		Pocillopora acuta		too low		too low
20250925		POC_R3_C3		Pocillopora acuta		too low		too low

TapeStation

After the isolation, I run samples on the tapestation, protocol linked above. See full report here.

TapeStation date		sample_id		TapeStation conc. ng/uL
20250925		B8		0.574
20250925		C10		0.71
20250925		C9		0.393
20250925		R12		0.42
20250925		R1		0.389
20250925		R3		0.473